Processes for reproducing sugar nucleotides and complex carbohydrates

ABSTRACT

This invention relates to a process for producing a sugar nucleotide, in which a) a culture broth of a microorganism capable of producing NTP from a nucleotide precursor, or a treated product of the culture broth, and b) a culture broth of a microorganism capable of producing a sugar nucleotide from a sugar and NTP, or a treated product of the culture broth, are used as enzyme sources; a process for producing a complex carbohydrate, in which the above-described a) and b) and c) a culture broth of a microorganism, an animal cell or an insect cell capable of producing a complex carbohydrate from a sugar nucleotide and a complex carbohydrate precursor, or a treated product of the culture broth, are used as enzyme sources; a process for producing a complex carbohydrate, in which a culture broth of a microorganism, an animal cell or an insect cell capable of producing a complex carbohydrate from a sugar nucleotide and a complex carbohydrate precursor, or a treated product of the culture broth, is as an enzyme source; and a process for producing N-acetylglucosamine-1-phosphate, in which a culture broth of a microorganism having strong galactokinase activity, or a treated product of the culture broth, is used as the enzyme source.

This application is a CPA filed Sep. 13, 2001 under 37 C F R §153(d)based on parent application Ser. No. 09/068,528 filed May 13, 1998.

TECHNICAL FIELD

This invention relates to a process for producing a complex carbohydratewhich is useful for protection against infection of bacteria, virusesand the like, application to cardiovascular disorders and immunotherapyand to a process for producing a sugar nucleotide which is important asa substrate for the synthesis of the complex carbohydrate.

BACKGROUND ART

Examples of the known process for producing sugar nucleotidesincludes: 1) chemical synthetic processes (Adv. Carbohydr. Chem.Biochem., 28, 307 (1973), Bull. Chem. Soc. Japan, 46, 3275 (1973), J.Org. Chem., 57, 146 (1992), Carbohydr. Res., 242, 69 (1993)); 2)production processes using enzymes (J. Org. Chem., 55, 1834 (1990), J.Org. Chem., 57, 152 (1992), J. Am. Chem. Soc., 110, 7159 (1988),Japanese Published Unexamined National Publication No. 508413/95,Japanese Published National Publication No. 500248/95, WO 96/27670); 3)processes using microbial cells such as yeast and the like (JapanesePublished Examined Patent Application No. 2073/70, Japanese PublishedExamined Patent Application No. 40756/71, Japanese Published ExaminedPatent Application No. 1837/72, Japanese Published Examined PatentApplication No. 26703/72, Japanese Published Examined Patent ApplicationNo. 8278/74, Japanese Published Unexamined Patent Application No.268692/90); and 4) an extraction process from microbial cells ofhalo-tolerant yeast (Japanese Published Unexamined Patent ApplicationNo. 23993/96).

However, the process 1) requires expensive materials (for example,morpholidate derivative of nucleotide-5′-monophosphate (referred to as“NMP” hereinafter), sugar phosphate, etc.); the process 2) requiresexpensive materials (for example, nucleotide-5′-diphosphate (referred toas “NDP” hereinafter), nucleotide-5′-triphosphate (referred to as “NTP”hereinafter), phosphoenolpyruvate, etc.), and various enzymes (e.g.,pyruvate kinase, etc.); and the process 3) requires drying treatment ofmicrobial cells. Including the process 4), all of the above-mentionedprocesses use expensive nucleotides, sugar phosphates, and the like orhave a difficulty in affecting large scale production from theoperational point of view, so that an industrial scale productionprocess of sugar nucleotides has not so far been established.

Examples of the known process for producing complex carbohydratesinclude 1) chemical synthetic processes (Method in Enzymol., 247, 193(1994), Angew. Chem. Int. Ed. Engl., 21, 155 (1982), Carbohydr. Res.,211, cl (1991)), 2) processes in which a hydrolase is used (Anal.Biochem., 202, 215 (1992), Trends Biotechnol., 6, 256 (1988)) and 3)processes in which a glycosyltransferase is used (Japanese PublishedUnexamined Patent Application No. 79792/95, Japanese Published NationalPublication No. 500248/95, Japanese Published Examined PatentApplication No. 82200/93, WO 94/25614, Japanese Published NationalPublication No. 503905/97, U.S. Pat. No. 5,583,042).

The introduction of protecting groups is essential for stereo-selectivesynthesis in the process 1). The yield and selectivity are notsufficient in the process 2). Expensive materials (for example, NDP,NTP, phosphoenolpyruvic acid, sugar phosphate, sugar nucleotide, etc.)and various enzymes (for example, pyruvate kinase, etc.) are necessaryin the process 3). Therefore, these processes have not been establishedas inexpensive industrial production processes of complex carbohydrates.In addition, there has been nothing known about a process for the directindustrial production of complex carbohydrates, which uses onlyinexpensive nucleotide precursors, sugars and complex carbohydrateprecursors as the starting materials.

It has been reported that UMP is produced in a microorganism belongingto the genus Corynebacterium when orotic acid is added (Amino Acid,Nucleic Acid, 23, 107(1971)). In addition, a process in which cytidinediphosphate choline is formed from orotic acid as the material is alsoknown (Japanese Published Unexamined Patent Application No. 276974/93).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a process for producinga complex carbohydrate which is useful for protection against infectionof bacteria, viruses, and the like, application to cardiovasculardisorders and immunotherapy, and a process for producing a sugarnucleotide which is important as a substrate for synthesizing thecomplex carbohydrate at a low cost and efficiently.

The inventors of the present invention have conducted intensive studieson the microbial production of a complex carbohydrate and a sugarnucleotide using a nucleotide precursor as the starting material, andhave found as the results that a sugar nucleotide can be produced byusing only a nucleotide precursor and a sugar as the materials, thatproductivity of the sugar nucleotide can be improved by strengtheningexpression of genes involved in its biosynthesis and that a complexcarbohydrate can be produced efficiently, using a microorganism capableof producing the sugar nucleotide and a microorganism, an animal cell oran insect cell capable of producing the complex carbohydrate from asugar nucleotide and a complex carbohydrate precursor and using anucleotide precursor, a sugar and a complex carbohydrate precursor asthe only starting materials, thereby resulting in the accomplishment ofthe present invention.

The present invention provides a process for producing a complexcarbohydrate, which comprises: selecting, as enzyme sources, a) aculture broth of a microorganism capable of producing NTP from anucleotide precursor, or a treated product of the culture broth, b) aculture broth of a microorganism capable of producing a sugar nucleotidefrom a sugar and NTP, or a treated product of the culture broth, and c)a culture broth of a microorganism, an animal cell or an insect cellcapable of producing a complex carbohydrate from a sugar nucleotide anda complex carbohydrate precursor, or a treated product of the culturebroth; allowing the enzyme sources, the nucleotide precursor, the sugarand the complex carbohydrate precursor to be present in an aqueousmedium to form and accumulate the complex carbohydrate in the aqueousmedium; and recovering the complex carbohydrate from the aqueous medium,a process for producing a sugar nucleotide, which comprises: selecting,as enzyme sources, a) a culture broth of a microoganism capable ofproducing NTP from a nucleotide precursor, or a treated product of theculture broth, and b) a culture broth of a microorganism capable ofproducing a sugar nucleotide from a sugar and NTP, or a treated productof the culture broth; allowing the enzyme sources, the nucleotideprecursor and the sugar to be present in an aqueous medium to form andaccumulate the sugar nucleotide in the aqueous medium; and recoveringthe sugar nucleotide from the aqueous medium, and a process forproducing a complex carbohydrate, which comprises: selecting, as anenzyme source, a culture bloth of a microorganism, an animal cell or aninsect cell capable of producing a complex carbohydrate from a sugarnucleotide and a complex carbohydrate precursor, or a treated product ofthe culture broth; allowing the enzyme source, the sugar nucleotideobtained by the above-described process for producing a sugar nucleotideand the complex carbohydrate precursor to be present in and aqueousmedium to form and accumulate the complex carbohydrate in the aqueousmedium; and recovering the complex carbohydrate from the aqueous medium.It also provides a process for producingN-acetylglucosamine-1-phosphate, which comprises selecting, as an enzymesource, a culture broth of a microorganism having strong galactokinaseactivity, or a treated product of the culture broth; allowing the enzymesource and N-acetylglucosamine to be present in an aqueous medium toform and accumulate N-acetylglucosamine-1-phosphate in the aqueousmedium; and recovering the N-acetylglucosamine 1-phosphate from theaqueous medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction steps of expression plasmids pPA31 and pPAC31.

FIG. 2 shows construction steps of galU, ppa gene expression plasmidspNT12 and pNT32.

FIG. 3 shows construction steps of galT, galK gene expression plasmidpNT25.

FIG. 4 shows construction steps of plasmid pTR7 which expresses galT andgalK genes in Corynebacterium ammoniagenes.

FIG. 5 shows construction steps of glmU, ppa gene expression plasmidpNT14.

FIG. 6 shows construction steps of pgm gene expression plasmid pNT24.

FIG. 7 shows construction steps of glmM gene expression plasmid pNT44.

FIG. 8 shows construction steps of glk gene expression plasmid pNT46.

FIG. 9 shows construction steps of pfkB gene expression plasmid pNT47.

FIG. 10 shows construction steps of galk gene expression plasmid pNT54.

FIG. 11 shows construction steps of manB, manC gene expression plasmidpNK7.

FIG. 12 shows construction steps of pgm, pfkB gene expression plasmidpNT55.

FIG. 13 shows construction steps of gmd, wcaG gene expression plasmidpNK8.

FIG. 14 shows construction steps of neuA gene expression plasmid pTA14.

FIG. 15 shows construction steps of lgtc gene expression plasmid pGT3.

FIG. 16 shows construction steps of lgtB gene expression plasmid pNT60.

Abbreviations to be used herein and description of the abbreviations areshown in Table 1-(1) and Table 1-(2).

TABLE 1 Glc glucose G-6-P glucose-6-phosphate G-1-P glucose-1-phosphateGlc-1, 6-P2 glucose-1, 6-diphosphate Gal galactose Gal-1-Pgalactose-1-phosphate GlcN-6-P glucosamine-6-phosphate GlcN-1-Pglucosamine-1-phosphate GlcUA glucuronic acid GlcN glucosamine GlcNAcN-acetylglucosamine GlcNAc-1-P N-acetylglucosamine-1-phosphate F-6-Pfructose-6-phosphate F-1, 6-P2 fructose-1,6-diphosphate Man mannoseMan-6-P mannose-6-phosphate Man-1-P mannose-1-phosphateGDP-4-keto-6-deoxyMan guanosine-5′-diphospho- 4-keto-6-deoxymannoseManNAc N-acetylmannosamine NeuAc N-acetylneuraminic acid acetyl CoAacetyl coenzyme A NTP nucleotide-5′-triphosphate NDPnucleotide-5′diphosphate NMP nucleotide-5′-monophosphate ATPadenosine-5′-triphosphate UTP uridine-5′-triphosphate GTPguanosine-5′-triphosphate CTP cytidine-5′-triphosphate GMPguanosine-5′-monophosphate UDP-G1c uridine-5′-diphosphoglucose UDP-Galuridine-5′-diphosphogalactose UDP-GlcNAcuridine-5′-diphospho-N-acetylglucosamine UDP-GalNAcuridine-5′-diphospho-N-acetylgalactosamine UDP-GlcUAuridine-5′-diphosphoglucuronic acid GDP-Manguanosine-5′-diphosphomannose GDP-Fuc guanosine-5′-diphosphofucoseCMP-NeuAc cytidine-5′-monophospho-N-acetylneuraminic acid galUglucose-1-phosphate uridyltransferase ppa (inorganic)pyrophosphatasegalK galactokinase galT galactose-1-phosphate uridyltransferase glmUN-acetylglucosamine-1-phosphate uridyltransferaseglucosamine-1-phosphate acetyltransferase pgm phosphoglucomutase pfkBphosphofructokinase glmM phosphoglucosamine mutase glk glucokinase manBphosphomannomutase manC mannose-1-phosphate guanyltransferase gmdGDP-mannose-4,6-dehydratase wcaG GDP-4-keto-6-deoxymannose epimerase/reductase neuA CMP-N-acetylneuraminic acid synthetase neuBN-acetylneuraminic acid synthase nanA N-acetylneuraminic acid aldolasepyrG cytidine-5′-triphosphate synthetase lgtB β1,4-galactosyltransferase1gtC α1,4-galactosyltransferase ugd UDP-glucose dehydrogenase

According to the present invention, a novel production process of asugar nucleotide and a novel production process of a complexcarbohydrate using the sugar nucleotide production process can beprovided, which are characterized in that 1) expensive materials (forexample, NTP, sugar phosphates, etc.) are not required, and inexpensivenucleotide precursor and a sugar can be used as the sole startingmaterials, 2) addition of expensive phosphoenolpyruvic acid and pyruvatekinase is not necessary in converting NMP or NDP into NTP, and 3) aprocess for the isolation of enzymes is not necessary.

With regard to the sugar nucleotide to be produced by the productionprocess of the present invention, compounds having a general structurein which the terminal phosphate group of a nucleotide 5′-diphosphateresidue and the reducing group of a sugar residue are linked together byester bonding can be exemplified, and those compounds in which thenucleotide residue is cytidine 5′-monophosphate and the sugar residue isa polyol are also included in the sugar nucleotide to be produced by thepresent invention.

Examples of the complex carbohydrate to be produced by the productionprocess of the present invention include compounds in whichcarbohydrates are bound to monosaccharides, oligosaccharides,monosaccharides or oligosaccharides linked to a carrier or the like,proteins, peptides, lipids, glycoproteins, glycolipids, glycopeptides,steroid compounds or the like.

The present invention will be described in detail below.

1) With regard to the microorganism for use in the present inventioncapable of producing NTP from a nucleotide precursor, any microorganismcapable of producing NTP from a nucleotide precursor can be used.Examples include microorganisms belonging to the genus Escherichia andthe genus Corynebacterium.

The microorganisms belonging to the genus Escherichia includeEscherichia coli and the like.

The microorganisms belonging to the genus Corynebacterium includeCorynebacterium ammoniagenes and the like.

2) As the microorganism for use in the present invention capable ofproducing a sugar nucleotide from a sugar and NTP, any microorganismhaving the activity to form the sugar nucleotide of interest can be usedas follows.

2)-(i) With regard to the production of UDP-Glc, it is preferred to usea microorganism having strong enzyme activities of (1) to (4) shown inthe following formula 1.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynebacterium can be exemplified. Specific examples includeEscherichia coli and Corynebacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (1), (2), (3) and (4) are increased byrecombinant DNA techniques can also be used. Specific examples of thetransformant include Escherichia coli KY8415 (FERM BP-408) havingrecombinant DNA (pNT12) which contains galU and ppa genes derived fromEscherichia coli, and the like.

(1): Hexokinase (EC 2.7.1.1) or glucokinase (EC 2.7.1.2)

(2): Phosphoglucomutase (EC 2.7.5.1)

(3): Glucose-1-phosphate uridyltransferase (EC 2.7.7.9)

(4): (Inorganic) pyrophosphatase (EC 3.6.1.1)

2)-(ii) With regard to the production of UDP-Gal, it is preferred to usea microorganism having strong enzyme activities of (5) and (6) shown inthe following formula 2, preferably further having strong enzymeactivities of (1) to (4) shown in the above-mentioned formula 1.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynebacterium can be exemplified. Specific examples includeEscherichia coli and Corynebacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (5) and (6), or at least one of the enzymeactivities selected from (5) and (6) and at least one of the enzymeactivities selected from (1) to (4), are increased by recombinant DNAtechniques can also be used. Specific examples of the transformantinclude Escherichia coli NM522 having recombinant DNA (pNT25) whichcontains galT and galK genes derived from Escherichia coli andCorynebacterium ammoniagenes ATCC 21170 having recombinant DNA (pTK7)which contains galT and galK genes derived from Escherichia coli.

(5): Galactokinase (EC 2.7.1.6)

(6): Galactose-1-phosphate uridyltransferase (EC 2.7.7.12)

2)-(iii) With regard to the production of UDP-GlcNAc, it is preferred touse a microorganism having strong enzyme activities of (7) to (12) shownin the following formula 3 and having a strong enzyme activity of (4)shown in formula 1, or a microorganism having strong enzyme activitiesof (13) and (10) shown in formula 3.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynebacterium can be exemplified. Specific examples includeEscherichia coli and Corynebacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (4), (7), (8), (9), (10) and (13) are increasedby recombinant DNA techniques can also be used. Specific examples of thetransformant include Escherichia coli NM522 having recombinant DNA(pNT44) which contains galmM gene derived from Escherichia coli,Escherichia coli KY8415 having recombinant DNA (pNT14) which containsglmU and ppa genes derived from Escherichia coli, Escherichia coli NM522having recombinant DNA (pNT46) which contains glk gene derived fromEscherichia coli, Escherichia coli NM522 having recombinant DNA (pNT54)which contains galK gene derived from Escherichia coli, and the like.

Although it is necessary to add Glc-1,6-P2 for the expression andincrement of the phosphoglucosamine mutase activity of (8) (J. Biol.Chem., 271, 32 (1996)), it is possible to provide Glc-1,6-P2 from G-6-Pand F-6-P without adding Glc-1,6-P2 by using a transformant in which theenzyme activities of (11) and (12) are increased by recombinant DNAtechniques.

Specific examples of such a transformant include Escherichia coli NM522having recombinant DNA (pNT24) which contains pgm gene derived fromEscherichia coli, Escherichia coli NM522 having recombinant DNA (pNT47)which contains pfkB gene derived from Escherichia coli, Escherichia coliNM522 having recombinant DNA (pNT55) which contains pgm and pfkB genederived from Escherichia coli, and the like.

The process in which expression of the phosphoglucosamine mutaseactivity of (8) is increased by providing Glc-1,6-P2 from G-6-P andF-6-P using the enzyme activities of (11) and (12) is a processdisclosed for the first time by the present invention.

The process in which GlcNAc-1-P is produced from GlcNAc using thegalactokinase (EC 2.7.1.6) of (13) is a process disclosed for the firsttime by the present invention. It is possible to produce GlcNAc-1-P byusing the process. That is, GlcNAc-1-P can be produced by using aculture broth or a treated product of the culture broth of amicroorganism having strong galactokinase activity, such as amicroorganism which contains recombinant DNA consisting of a DMAfragment containing a galK-encoding gene and a vector, as an enzymesource, allowing the enzyme source and GlcNAc to be present in anaqueous medium to form and accumulate GlcNAc-1-P in the aqueous medium,and recovering the thus-obtained GlcNAc-1-P from the aqueous medium.

Recovery of GlcNAc-1-P from the aqueous medium can be carried out in theusual way in which activated carbon, an ion exchange resin, and the likeare used.

(7): Hexokinase (EC 2.7.1.1) or glucokinase (EC 2.7.1.2)

(8): Phosphoglucosamine mutase

(9): Glucosamine-1-phosphate acetyltransferase

(10): N-Acetylglucosamine-1-phosphate uridyltransferase (EC 2.7.7.23)

(11): Phosphofructokinase (EC 2.7.1.11)

(12): Phosphoglucomutase (EC 2.7.5.1)

(13): Galactokinase (EC 2.7.1.6)

2)-(iv) With regard to the production of UDP-GalNAc, it is preferred touse a microorganism having strong enzyme activities of (7) to (12) shownin formula 3, of (14) shown in formula 4 and of (4) shown in formula 1,or a microorganism having strong enzyme activities of (10) and (13)shown in formula 4 and of (14) shown in formula 4.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynabacterium can be exemplified. Specific examples includeEscherichia coli and Corynabacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (7) to (14) and (4) are increased byrecombinant DNA techniques can also be used.

(14) UDP-GlcNAc 4-epimerase (EC 5.1.3.7)

2)-(v) With regard to the production of UDP-GlcUA, it is preferred touse a microorganism having strong enzyme activities of (1) to (4) shownin formula 1 and of (15) shown in formula 5.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynabacterium can be exemplified. Specific examples includeEscherichia coli and Corynabacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (1), (2), (3), (4) and (15) are increased byrecombinant DNA techniques can also be used.

(15): UDP-Glc dehydrogenase (EC 1.1.1.22)

2)-(vi) With regard to the production of GDP-Man, it is preferred to usea microorganism having strong enzyme activities of (16) to (18) shown inthe following formula 6 and of (11) and (12) shown in formula 3.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynabacterium can be exemplified. Specific examples includeEscherichia coli and Corynabacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (16), (17) and (18) are increased byrecombinant DNA techniques can also be used. Specific examples of thetransformant include Escherichia coli NM522 having recombinant DNA(pNK7) which contains manB and manC genes derived from Escherichia coli,Escherichia coli NM522 having recombinant DNA (pNT46) which contains glkgene derived from Escherichia coli, and the like.

Although it is necessary to add Glc-1,6-P2 for the expression andincrement of the phosphomannomutase activity of (17) by recombinant DNAtechniques, it is possible to provide Glc-1,6-P2 from G-6-P and F-6-Pwithout adding Glc-1,6-P2 by using a transformant in which the enzymeactivities of (11) and (12) are increased by recombinant DNA techniques.Specific examples of such a transformant include Escherichia coli NM522having recombinant DNA (pNT24) which contains pgm gene derived fromEscherichia coli, Escherichia coli NM522 having recombinant DNA (pNT47)which contains pfkB gene derived from Escherichia coli, Escherichia coliNM522 having recombinant DNA (pNT55) which contains pgm and pfkB genesderived from Escherichia coli, and the like.

The process in which expression of the phosphomannomutase activity of(17) is increased by providing Glc-1,6-P2 from G-6-P and F-6-P using theenzyme activities of (11) and (12) is a process disclosed for the firsttime by the present invention.

(16): Hexokinase (EC 2.7.1.1) or glucokinase (EC 2.7.1.2)

(17): Phosphomannomutase (EC 2.7.5.7)

(18): Mannose-1-phosphate guanyltransferase (EC 2.7.7.13)

2)-(vii) With regard to the production of GDP-Fuc, it in preferred touse a microorganism having strong enzyme activities of (19) and (20)shown in the following formula 7, of (16) to (18) shown in formula 6 andof (11) and (12) shown in formula 3.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynabacterium can be exemplified. Specific examples includeEscherichia coli and Corynabacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (16), (17), (18), (19) and (20) are increasedby recombinant DNA techniques can also be used. Specific examples of thetransformant include Escherichia coli NM522 having recombinant DNA(pNK7) which contains manB and manC genes derived from Escherichia coli,Escherichia coli NM522 having recombinant DNA (pNK8) which contains gmdand wcaG genes derived from Escherichia coli, Escherichia coli NM522having recombinant DNA (pNT46) which contains glk gene derived fromEscherichia coli, and the like.

Although it is necessary to add Glc-1,6-P2 for the expression andincrement of the phosphomannomutase activity of (17) by recombinant DNAtechniques, it is possible to provide Glc-1,6-P2 from G-6-P and F-6-Pwithout adding Glc-1,6-P2 by using a transformant in which the enzymeactivities of (11) and (12) are increased by recombinant DNA techniques.

Specific examples of such a transformant include Escherichia coli NM522having recombinant DNA (pNT24) which contains pgm gene derived fromEscherichia coli, Escherichia coli NM522 having recombinant DNA (pNT47)which contains pfkB gene derived from Escherichia coli, Escherichia coliNM522 having recombinant DNA (pNT55) which contains pgm and pfkB genesderived from Escherichia coli, and the like.

(19): GDP-Man-4,6-dehydratase (EC 4.2.1.47)

(20): GDP-4-keto-6-deoxymannose epimerase/reductase

2)-(viii) With regard to the production of CMP-NeuAc, it is preferred touse a microorganism having strong enzyme activities of (21), (22) or(23), (24) and (25) shown in the following formula 8.

Specifically, microorganisms belonging to the genus Escherichia and thegenus Corynabacterium can be exemplified. Specific examples includeEscherichia coli and Corynabacterium ammoniagenes.

In addition, a transformant in which at least one of the enzymeactivities selected from (21), (22), (23), (24) and (25) are increasedby recombinant DNA techniques can also be used. Specific examples of thetransformant include Escherichia coli C600 having recombinant DNA(pNAL1) which contains nanA gene derived from Escherichia coli (Appl.Environ. Microbiol., 51, 562 (1986)), Escherichia coli NM522 havingrecombinant DNA (pTA14) which contains neuA gene derived fromEscherichia coli, and the like

(21): GlcNAc 2-epimarase (EC 5.1.3.8)

(22): NeuAc aldolase (EC 4.1.3.3)

(23): NeuAc synthetase (EC 4.1.3.19)

(24): CMP-NeuAc synthetase (EC 2.7.7.43)

(25): CTP synthetase (EC 6.3.4.2)

When a microorganism has both of the properties of microorganismsdescribed in 1) and the properties of microorganisms described in 2), itis possible to produce a sugar nucleotide from a nucleotide precursorand a sugar using the microorganism.

It is possible to produce UDP-Glc from a UTP precursor such as oroticacid or the like and glucose using a microorganism which has both of theproperties of microorganisms described in 1) and the properties ofmicroorganisms described in 2)-(i); UDP-Gal from a UTP precursor such asorotic acid or the like and galactose using a microorganism which hasboth of the properties of microorganisms described in 1) and theproperties of microorganisms described in 2)-(ii); UDP-GlcNAc from a UTPprecursor such as orotic acid or the like and glucosamine orN-acetylglucosamine using a microorganism which has both of theproperties of microorganism descried in 1) and the properties ofmicroorganisms described in 2)-(iii); UDP-GalNAc from a UTP precursorsuch as orotic acid or the like and glucosamine or N-acetylglucosamineusing a microorganism which has both of the properties of microorganismsdescribed in 1) and the properties of microorganisms described in2)-(iv); UDP-GlcUA from a UTP precursor such as orotic acid or the likeand glucose using a microorganism which has both of the properties ofmicroorganisms described in 1) and the properties of microorganismsdescribed in 2)-(v); GDP-Man from a GTP precursor such as GMP or thelike and mannose using a microorganism which has both of the propertiesof microorganisms described in 1) and the properties of microorganismsdescribed in 2)-(vi); GDP-Fuc from a GTP precursor such as GMP or thelike and mannose using a microorganism which has both of the propertiesof microorganism described in 1) and the properties of microorganismdescribed in 2)-(vii); and CMP-NeuAc from a CTP precursor such as oroticacid or the like and N-acetylglucosamine or N-acetylmannosamine using amicroorganism which has both of the properties of microorganismsdescribed in 1) and the properties of microorganism described in2)-(viii).

Specific examples of such microorganism include Corynabacteriumammoniagenes capable of expressing galT and galK genes derived fromEscherichia coli.

Unlike the case of the above-mentioned strain, when a single strain hasonly a part of activities required for producing a sugar nucleotide, thesugar nucleotide can be produced by optionally combining microorganismshaving respective activities.

The properties described in 1) are not necessarily owned by a singlemicroorganism, and two or more microorganisms in which the propertiesdescribed in 1) are independently located can also be used as themicroorganism having the properties described in 1). Specifically, acombination of Escherichia coli capable of expressing Escherichiacoli-derived pyrG gene with Corynabacterium ammoniagenes (JapanesePublished Unexamined Patent Application No. 276974/93) is exemplified.

In the same manner, the microorganism having the properties described in2) is not necessarily a single microorganism and the properties canindependently be owned by two or more microorganisms. By optionallycombining the microorganisms, each sugar nucleotide of interest can beproduced.

For example, it is possible to produce UDP-Glc from a UTP precursor suchas orotic acid or the like and glucose using a microorganism which hasthe properties of microorganism described in 1) and at least onemicroorganism having the properties described in 2)-(i); UDP-Gal from aUTP precursor such as orotic acid or the like and galactose using amicroorganism which has the properties of microorganisms described in 1)and at least one microorganism having the properties described in2)-(ii); UDP-Glc fr a UTP precursor such as orotic acid or the like andglucosamine or N-acetylglucosamine using a microorganism which has theproperties of microorganisms described in 1) and at least onemicroorganism having the properties described in 2)-(iii); UDP-GalNAcfrom a UTP precursor such as orotic acid or the like and glucosamine orN-acetylglucosamine using a microorganism which has the properties ofmicroorganisms described in 1) and at least one microorganisms havingthe properties described in 2)-(iv); UDP-GlcUA from a UTP precursor suchas orotic acid or the like and glucose using a microorganism which hasthe properties of microorganisms described in 1) and at least onemicroorganism having the properties described in 2)-(v); GDP-Man from aGTP precursor such as GMP or the like and mannose using a microorganismwhich has the properties described in 1) and at least one microorganismhaving the properties described in 2)-(vi); GDP-Fuc from a GTP precursorsuch as GMP or the like and mannose using a microorganism which has theproperties described in 1) and at least one microorganism having theproperties described in 2)-(vii); and CMP-NeuAc from a CTP precursorsuch as orotic acid or the like and N-acetylglucosamine orN-acetylmannosamine using a microorganism which has the propertiesdescribed in 1) and at least one microorganisms having the propertiesdescribed in 2)-(viii) .

As described in the foregoing, remaining recombinant microorganisms canbe used in the production of sugar nucleotides, and the genes shown inTable 2 related to the production of sugar nucleotides have been clonedfrom the chromosomes of Escherichia coli and their complete nucleotidesequences have been determined.

TABLE 2 Genes References galU gene J. Biochem, 115, 965 (1994) ppa geneJ. Bacteriol., 170, 5901 (1988) galK gene Nucleic Acids Res., 13, 1841(1985) galT gene Nucleic Acids Res., 14, 7705 (1986) glmU gene J.Bacteriol, 175, 6150 (1993) pgm gene J. Bacteriol, 176, 5847 (1994) pfkBgene Gene, 28, 337 (1984) glmM gene J. Biol. Chem., 271, 32 (1996) glkgene J. Bacteriol., 179, 1298 (1997) manB gene J. Bacteriol., 178, 4885(1996) manC gene J. Bacteriol., 178, 4885 (1996) gmd gene J. Bacteriol.,178, 4885 (1996) wcaG gene J. Bacteriol., 178, 4885 (1996) neuA gene J.Biol. Chem., 264, 14769 (1989) neuB gene J. Bacteriol., 177, 312 (1995)nanA gene Nucleic Acids Res., 13, 8843 (1985) pyrG gene J. Biol. Chem.,261, 5568 (1986) ugd gene J. Bacteriol., 177, 4562 (1995)

Various procedures related to recombinant DNA techniques, such asisolation and purification of plasmid DNA from Escherichia coli having aplasmid which contains the genes, cleavage of the plasmid DNA withrestriction enzymes, isolation and purification of the cleaved DNAfragments, enzymatic linking of the DNA fragments and transformation ofEscherichia coli using recombinant DNA, can be carried out in accordancewith known processes (for example, J. Sambrook et al., MolecularCloning, A Laboratory Manual, second edition, Cold Spring HarborLaboratory (1989)). In addition, the polymerase chain reaction (referredto as “PCR” hereinafter) can be carried out, for example, using ThermalCycler manufactured by Perkin-Elmer-Cetus.

Expression of a gene related to the production of a sugar nucleotide ina host can be affected by a procedure in which a fragment containing thegene is obtained as an appropriate length of DNA fragment containing thegene using restriction enzymes or PCR and then the thus formed DNAfragment is inserted into downstream of the promoter of an expressionvector, and the DNA-inserted expression vector is introduced into a hostcell which is suited for the expression vector.

Every microorganism can be used as the host, so long as it can expressthe gene of interest. Examples include microorganism belonging to thegenus Escherichia, Serratia, Corynabacterium, Brevibacterium,Pseudomonas, Bacillus and the like, as well as yeasts belonging to thegenus Saccharomyces, Candida and the like.

With regard to the expression vector to be used, those having an abilityto replicate autonomously in the above-described host or to beintegrated into chromosome, and containing a promoter at the position atwhich transcription of the gene related to the production of a sugarnucleotide can be affected, may be used.

When the above-mentioned microorganism is used as the host, it ispreferred that the expression vector of a gene related to the productionof a sugar nucleotide can be replicated autonomously in themicroorganism and that, at the same time, the expression vectorcomprises a promoter, a ribosome-binding sequence, the gene related tothe production of sugar nucleotide and a transcription terminatorsequence. It may also contain a regulatory gene of the promoter.

Examples of the expression vector include pBTrp2, pBTac1, pBTac2 (allmanufactured by Boehringer Manheim Co.), pKYP10 (Japanese PublishedUnexamined Patent Application No. 110600/83), pKYP200 (Agric. Biol.Chem., 48, 669 (1994)), pLSA1 (Agric. Biol. Chem., 53, 277 (1989)),pGEL1 (Proc. Natl. Acad. Sci. USA., 82, 4306 (1985)), pBluescript IISK+(manufactured by STRATAGENE), pTrS30 (prepared from Escherichia coliJM109/pTrS30 (FERM BP-5407)), pTrS32 (prepared from Escherichia coliJM109/pTrS32 (FERM BP-5408)), pUC19 (Gene, 33, 103 (1985)), pSTV28(manufactured by Takara Shuzo Co., Ltd.), pPA1 (Japanese PublishedUnexamined Patent Application No. 233798/88), pCG11(Japanese ExaminedPatent Application No. 91827/94), and the like.

Any promoter can be used, so long as it can be expressed in theabove-mentioned host. Examples include promoters derived fromEscherichia coli, phage and the like, such as trp promoter, lacpromoter, P_(L) promoter, P_(R) promoter, and the like. Also usable areartificially designed and modified promoters such as trp tandem promoterin which two trp promoters are connected in series, tac promoter, andthe like.

With regard to the ribosome-binding sequence, any sequence capable ofbeing expressed in the above-mentioned host can be used, but it ispreferred to use a plasmid in which the region between aribosome-binding sequence and an initiation codon is adjusted to asuitable distance (for example, 6 to 18 bases).

Although the transcription terminator sequence is not always necessaryfor the expression of genes related to the production of sugarnucleotide, it is preferred to arrange the transcription terminationsequence preferably at a downstream position of the structural gene.

Any microorganism can be used as the host, so long as it can express therecombinant DNA and can apply to the sugar nucleotide formationreaction. Examples include Escherichia coli XL1-Blue, Escherichia coliXL2-Blue, Escherichia coliDB1, Escherichia coli MC1000, Escherichia coliKY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coliHB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coliNY49, Escherichia coli KY8415, Escherichia coli NM522, Bacillussubtilis, Bacillus brevis, Bacillus amyloliquefaciens, Brevibacteriumimmariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066,Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC13869, Corynabacterium ammoniagenes ATCC 21170, Corynabacteriumglutamicus ATCC 13032, Corynabacterium acetoacidophilum ATCC 13870,Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida, Serratiamarcescens, and the like.

When a yeast strain is used as the host, the expression vector may beYEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419) or the like.

With regard to the promoter, any promoter capable of being expressed inthe yeast strain host can be used. Examples include promoters in genesof the glycolytic pathway, such as hexokinase as well as other promoterssuch as gal 1 promoter, gal 10 promoter, heat shock protein promoter,MFα1 promoter and CUP 1 promoter.

With regard to the host, any yeast capable of expressing recombinant DNAand applying to the sugar nucleotide formation reaction can be used.Examples include Saccharomyces cerevisiae, Candida utilis, Candidaparapsilosis, Candida krusei, Candida versatilis, Candida lipolytica,Candida zeylanoides, Candida guilliermondii, Candida albicans, Candidahumicola, Pichia farinosa, Pichia ohmeri, Torulopsis candida, Torulopsissphaerica, Torulopsis xylinus, Torulopsis famata, Torulopsis versatilis,Debaryomyces subglobosus, Debaryomyces cantarellii, Debaryomycesglobosus, Debaryomyces hansenii, Debaryomyces japonicus,Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Kluyveromyceslactis, Kluyveromyces marxianus, Hansenula anomala, Hansenula jadinii,Brettanomyces lambicus, Brettanomyces anomalus, Schizosaccharomycespombe, Trichosporon pullulans, Schwanniomyces alluvius, and the like.

Culturing of the microorganism for use in the present invention can becarried out in accordance with the usual culturing process. The mediumfor use in the culturing of the microorganism may be either a nutrientmedium or a synthetic medium, so long as it contains carbon sources,nitrogen sources, inorganic salts and the like which can be assimilatedby the microorganism and it can perform culturing of the microorganismefficiently.

Examples of the carbon sources include those which can be assimilated byeach microorganism, such as carbohydrates (for example, glucose,fructose, sucrose, lactose, maltose, mannitol, sorbitol, molasses,starch, starch hydrolysate, etc.), organic acids (for example, pyruvicacid, lactic acid, citric acid, fumaric acid, etc.), various amino acids(for example, glutamic acid, methionine, lysine, etc.), and alcohols(for example, ethanol, propanol, glycerol, etc.). Also useful arenatural organic nutrient sources, such as rice bran, cassava, bagasse,corn steep liquor, and the like.

Examples of the nitrogen sources include various inorganic and organicammonium salts (for example, ammonia, ammonium chloride, ammoniumsulfate, ammonium carbonate, ammonium acetate, ammonium phosphate,etc.), amino acids (for example, glutamic acid, glutamine, methionine,etc.), peptone, NZ amine, corn steep liquor, meat extract, yeastextract, malt extract, casein hydrolysate, soybean meal, fish meal or ahydrolysate thereof and the like.

Examples of the inorganic substances include potassium dihydrogenphosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate,disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate,magnesium chloride, sodium chloride, calcium chloride, ferrous sulfate,manganese sulfate, copper sulfate, zinc sulfate, calcium carbonate, andthe like. Vitamins, amino acids, nucleic acids and the like may be addedas occasion demands.

The culturing is carried out under aerobic conditions by shakingculture, aeration stirring culture or the like means. The culturingtemperature is preferably from 15 to 45° C., and the culturing time isgenerally from 5 to 96 hours. The pH of the medium is maintained at 3.0to 9.0 during the culturing. Adjustment of the medium pH is carried outusing an inorganic or organic acid, an alkali solution, urea, calciumcarbonate, ammonia and the like. Also, antibiotics (for example,ampicillin, tetraycycline, etc.) may be added to the medium during theculturing as occasion demands.

When a microorganism transformed with an expression vector in which aninducible promoter is used as the promoter is cultured, an inducer maybe to the medium as occasion demands. For example,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added tothe medium when a microorganism transformed with an expression vectorcontaining lac promoter is cultured, or indoleacrylic acid (IAA) or thelike may by added thereto when a microorganism transformed with anexpression vector containing trp promoter is cultured.

When two or more microorganisms are used in the sugar nucleotideproduction of the present invention, the microorganisms may beseparately cultured to use the resulting culture broths in the sugarnucleotide production or inoculated simultaneously into a single culturevessel to carry out mixed culturing and to use the resulting culturebroth in the sugar nucleotide production. In an alternative way, one ofthe microorganisms is firstly cultured, the remaining microorganism isinoculated during or after the culturing and cultured, and then theresulting culture broth is used in the sugar nucleotide production. Inanother alternative way, a microorganism having the properties describedin 1) and a microorganism having the properties described in 2) may beseparately culture and used in the sugar nucleotide production using theresulting culture broths.

The microbial culture broth obtained by the culturing or a treatedproduct of the culture broth obtained by treating the culture broth invarious ways can be used as an enzyme source for the formation of asugar nucleotide in an aqueous medium.

Examples of the treated product of the culture broth include aconcentrated product of the culture broth, a dried product of theculture broth, a culture supernatant obtained by centrifuging theculture broth, a concentrated product of the culture supernatant, anenzyme preparation obtained from the culture supernatant, cells(including microbial cells) obtained by centrifuging the culture broth,a dried product of the cells, a freeze-dried product of the cells, asurfactant-treated product of the cells, a mechanically disruptedproduct of the cells, a solvent-treated product of the cells, anenzyme-treated product of the cells, a protein fraction of the cells, animmobilized product of the cells and an enzyme preparation obtained byextraction from the cells.

The amount of the enzyme source used in the formation of the sugarnucleotide is within the range of from 1 to 500 g/l, preferably from 5to 300 g/l, as wet cells. When the reaction is carried out in an aqueousmedium using two or more microorganisms simultaneously, amount of thetotal wet cells of the microorganisms in the aqueous medium is withinthe range of from 2 to 500 g/l, preferably from 5 to 400 g/l.

Examples of the aqueous medium used in the formation of the sugarnucleotide include water, buffer solutions (for example, those ofphosphate, carbonate, acetate, borate, citrate, Tris, etc.), alcohols(for example, methanol, ethanol, etc.), and the like. The microbialculture broth used as the enzyme source may also be used as the aqueousmedium.

Examples of the nucleotide precursor used in the formation of the sugarnucleotide include orotic acid, uracil, orotidine, uridine, cytosine,cytidine, adenine, adenosine, guanine, guanosine, hypoxanthine, inosine,xanthine, xanthosine, inosine-5′-monophospate,xanthosine-5′-monophosphate, guanosine-5′-monophosphate,uridine-5′-monophosphate, cytidine-5′-monophosphate, and the like.Preferred are orotic acid and guanosine-5′-monophosphate. The nucleotideprecursor may be in the form of a purified product or in the form of asalt of the precursor, and a culture broth containing the precursorproduct by the formation of a microorganism or the precursor roughlypurified from the culture broth may also be used as the nucleotideprecursor, so long as its impurities do not inhibit the reaction. Thenucleotide precursor is used at a concentration of from 0.1 mM to 1.0 M,preferably from 0.01 to 0.3 M.

Examples of the sugar used in the formation of the sugar nucleotideinclude glucose, fructose, galactose, glucosamine, N-acetylglucosamine,N-acetylgalactosamine, mannose, fucose, N-acetylmannosamine,N-acetylneuraminic acid and the like, and derivatives thereof. The sugarmay be either in the form of a purified product or in the form of amaterial containing the same, so long as impurities in the material donot inhibit the reaction. The sugar is used at a concentration of from0.1 mM to 2.0 M, by adding it in one portion when the reaction isstarted or in portions or continuously during the reaction.

In the formation of the sugar nucleotide, an energy source necessary forthe regeneration of ATP, a coenzyme, a phosphate ion, a magnesium ion, achelating agent (for example, phytic acid, etc.), a surfactant and anorganic solvent may be added as occasion demands.

Examples of the energy source include carbohydrate (for example,glucose, fructose, sucrose, lactose, maltose, mannitol, sorbitol,molasses, starch hydrolysate etc.), organic acids (for example, pyrivicacid, lactic acid, acetic acid, etc.), amino acids (for example,glycine, alanine, aspartic acid, glutamic acid, etc.), and the like,which may be used at a concentration of from 1.0 mM to 2.0 M.

Examples of the phosphate ion include orthophosphoric acid,polyphosporic acids (for example, pyrophosphoric acid, tripolyphosphoricacid, tetrapolyphosphoric acid, tetrapolymetaphosphoric acid, etc.),polymetaphosphoric acids, inorganic phosphates (for example, potassiumdihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogenphosphate, disodium hydrogen phosphate, etc.), and the like, which maybe used at a concentration of from 1.0 mM to 1.0 M.

Examples of the magnesium ion include inorganic magnesium salts (forexample, magnesium sulfate, magnesium nitrate, magnesium chloride,etc.), organic magnesium salts (for example, magnesium citrate, etc.),and the like, which may be used at a concentration of generally from 1to 100 mM.

Examples of the surfactant include those which can enhance theproduction of various sugar nucleotides, such as nonionic surfactants(for example, polyoxyethylene octadecylamine (e.g., Nymean s-215,manufactured by Nippon Oils and Fats Co.), etc.), cationic surfactants(for example, catyl trimethylammonium bromide, alkyldimethylbenzylammonium chloride (e.g., Cation F2-40E, manufactured by NipponOils and Fats Co.) etc.) anionic surfactants (for example, lauroylsarcosinate, etc.) and tertiary amines (for example, alkyldimethylamine(e.g., Tertiary Amine FB, manufactured by Nippon Oils and Fats Co.),etc.) which may be used alone or as a mixture of two or more. Thesurfactant may be used at a concentration of generally from 0.1 to 50g/l.

Examples of the organic solvents include xylene, toluene, aliphaticalcohol, acetone, ethyl acetate, and the like, which may be used at aconcentration of generally from 0.1 to 50 ml/l.

The reaction for forming a sugar nucleotide can be carried out in anaqueous medium at pH of from 5 to 10, preferably from 6 to 9, at atemperature of from 20 to 50° C. and for a period of from 2 to 96 hours.

The sugar nucleotide can be formed by the process. Examples include auridine diphosphate compound, a guanosine diphosphate compound, acytidine monophosphate compound and the like. Specific examples includesugar nucleotides selected from UDP-Glc, UDP-Ga1, UDP-GlcNAc,UDP-Ga1NAc, UDP-GlcUA, GDP-Man, GDP-Fuc, CMP-NeuAc, and the like, andderivatives thereof.

Determination of the sugar nucleotide formed in the aqueous medium canbe carried out in accordance with a known method, for example, isolationand determination of UDF-Glc and UDP-Ga1 can be carried out by highperformance liquid chromatography (referred to as “HPLC” hereinafter)method described in Anal. Biochem., 216, 188 (1994). In addition,isolation and determination of UDP-GlcNAc, GDP-Man, GDP-Fuc andCMP-NeuAc cna be carried out by HPLC under the following conditions:

Elution Solution

0.1 M KH₂PO₄ (adjust to pH 3.2 with H₃PO₄)

Flow Rate

1 ml/min

Column

Partisil-10 SAX (manufactured by Whatman)

Detection

UV 262 nM

Determination

Calculated by comparing standard absorbance values

Recovery of the sugar nucleotide formed in the reaction solution can becarried out in the usual way using activated carbon, an ion exchangeresin and the like, for example, UDP-Ga1 and UDP-Glc can be recovered inaccordance with the process described in J. Org. Chem., 57, 152 (1992),and UDP-GlcNAc with the process described in J. Org. Chem., 57, 146(1992).

With regard to the microorganisms, animal cells or insect cells eligiblefor in the production of the complex carbohydrate of the presentinvention, all microorganisms, animal cells or insect cells capable ofproducing the complex carbohydrate from a sugar nucleotide and a complexcarbohydrate precursor can be used. Examples of such microorganisms,animal cells or insect cells include those which have the activities ofglucosyltransferase, galactosyltransferase,N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase,glucuronosyltransferase, mannosyltransferase, sialyltransferase,fucosyltransferase, and the like.

In addition, microorganisms, animal cells or insect cells modified byrecombinant DNA techniques can also be used in a similar way to the caseof the above-mentioned sugar nucleotide production. Examples of suchmicroorganisms, animal calls or insect cells include Escherichia coliwhich expresses the ceramide glucosyltransferase gene derived from humanmelanoma cell line SK-Mel-28 (Proc. Natl. Acad. Sci. USA., 93, 4638(1996)), human melanoma cell line WM266-4 which producesβ1,3-galacytosyltransferase (ATCC CRL 1676), recombinant cell line suchas namalwa cell line KJM-1 or the like which contains theβ1,3-galactosyltransferase gene derived from the human melanoma cellline WM266-4 (Japanese Published Unexamined Patent Application No.181759/94), Escherichia coli (EMBO J., 9, 3171 (1990)) or Saccharomycescerevisiae (Biochem, Biophys. Res. Commun., 201, 160 (1994)) whichexpresses the β1,4-galacytosyltransferase gene derived from human HeLacells, COS-7 cell line (ATCC CRL 1651) which expresses the ratβ1,6-N-acetylglucosaminyltransferase gene (J. Biol. Chem., 268, 15381(1993)), Sf9 cell line which expresses humanN-acetylglucosaminyltransferase gene (J. Biochem., 118, 568 (1995)),Escherichia coli which expresses human glucuronosyltransferase (Biochem.Biophys. Res. Commun., 196, 473 (1993)), namalwa cell line whichexpresses human α1,3-fucosyltransferase (J. Biol. Chem., 269, 14730(1994)), COS-1 cell line which expresses humanα1,3/1,4-fucosyltransferase (Genes Dev., 4, 1288 (1990)), COS-1 cellline which expresses human α1,2-fucosyltransferase (Proc. Natl. Acad,Sci. USA., 87, 6674 (1990)), COS-7 cell line which expresses chickenα2,6-sialyltransferase (Eur. J. Biochem., 219, 375 (1994)), COS cellline which expresses human α2,8-sialyltransferase (Proc. Natl. Acad.Sci. USA., 91, 7952 (1994)), Escherichia coli which expressesβ1,3-N-acetylglucosaminyltransferase, β1,4-galactosyltransferase,β1,3-N-acetylgalactosaminyltransferase or α1,4-galactosyltransferasederived from Neisseria (WO 96/10086), Escherichia coli which expressesNeisseria-derived α2,3-sialyltransferase (J. Biol. Chem., 271, 28271(1996)), Escherichia coli which expresses Helicobactar pylori-derivedα1,3-fucosyltransferase (J. Biol. Chem., 272, 21349 and 21357 (1997)),Escherichia coli which expresses yeast-derived α1,2-mannosyltransferase(J. Biol. Chem., 58, 3985 (1993)), and the like.

When a microorganism is used for producing the complex carbohydrate ofthe present invention, the microorganism can be cultured using the samemedium under the same culture conditions as in the case of theabove-mentioned microorganism capable of producing a sugar nucleotidefrom a nucleotide precursor and a sugar.

When animal cells are used for producing the complex the complexcarbohydrate of the present invention, the preferred culture medium isgenerally RPMI 1640 medium, Eagle's MEM medium or a medium thereofmodified by further adding fetal calf serum, and the like. The culturingis carried out under certain conditions, for example, in the presence of5% CO₂. The culturing is carried out at a temperature of preferably from20 to 40° C. for a period of generally from 3 to 14 days. As occasiondemands, antibiotics may be added to the medium.

When insect cells are used for producing the complex carbohydrates ofthe present invention, culturing of the insect cells can be carried outin accordance with the known process (J. Biol. Chem., 268, 12609(1993)).

The culture broth of a microorganism, an animal cell line or an insectcell line obtained by the culturing and a treated product of the culturebroth obtained by treating the culture broth in various ways can be usedas an enzyme source for forming the complex carbohydrate in an aqueousmedium.

Examples of the treated product of the culture broth include aconcentrated product of the culture broth, a dried product of theculture broth, a culture supernatant obtained by centrifuging theculture broth, a concentrated product of the culturesupernatant, anenzyme preparation obtained from the culture supernatant, cells(including microbial cells) obtained by centrifuging the culture broth,a dried product of the cells, a freeze-dried product of the cells, asurfactant-treated product of the cells, an ultrasonic-treated productof the cells, a mechanically disrupted product of the cells, asolvent-treated product of the cells, an enzyme-treated product of thecells, a protein fraction of the cells, an immobilized product of thecells and an enzyme preparation obtained by extraction from the cells.

The enzyme source used in the formation of the complex carbohydrate istypically within the range of from 0.1 mU/l to 10,000 U/l, preferablyfrom 1 mU/l to 1,000 U/l (where 1 units (U) is the amount of the enzymeactivity which can form 1 μmole of the complex carbohydrate within1minute at 37° C.).

Examples of the aqueous medium used in the formation of the complexcarbohydrate include water, buffer solutions (for example, those ofphosphate, carbonate, acetate, borate, citrate, Tris, etc.), alcohols(for example, methanol, ethanol, etc.), esters (for example, ethylacetate, etc.), ketones (for example, acetone, etc.), amides (forexample, acetamide, etc.), and the like. Each of the culture broths ofmicroorganisms, animal cells or insect cells used as the enzyme sourcemay also be used as the aqueous medium.

As occasion demands, chelating agents (for example, phytic acid, etc.),inorganic salts (for example, Mncl₂ , etc.), β-mercaptoethanol and thelike may be added.

As the sugar nucleotide used in the formation of the complexcarbohydrate, the above-mentioned reaction solution obtained by thesugar nucleotide formation or the sugar nucleotide purified from thereaction solution can be used at a concentration of from 0.01 mM to 2.0M.

In addition, a sugar nucleotide can be supplied in the complexcarbohydrate formation reaction solution by forming the sugar nucleotideby the above-mentioned process.

With regard to the complex carbohydrate precursor used in the formationof the complex carbohydrate, any material can be used, so long as it canbe used as the substrate of glycosyltransferase. Examples includemonosaccharides, oligosaccharides, monosaccharides or oligosaccharideslinked to a carrier or the like, proteins, peptides, lipids,glycoproteins, glycolipids, glycopeptides, steriod compounds, and thelike.

Specific examples include glucose, galactose, mannose, sialic acid,N-acetylglucosamine, N-acetylgalactosamine, lactose,N-acetyllactosamine, lacto-N-biose, GlcNAcβ1-3GAlβ1-4Glc,GlcNAcβ1-4Galβ1-4Glc, globotriose, Galα1-4Galβ1-4GlcNAc,2′-fucosyllactose, 3-fucosyllactose, 3′-sialyllactose, 6′-sialyllactose,3′-sialyl-N-acetyllactosamine, 6′-sialyl-N-acetyllactosamine,sialyllacto-N-biose, Lewis X, Lewis a, lacto-N-tetraose,lacto-N-neotetraose, lactodifucotetraose, 3′-sialyl-3-fucosyllactose,sialyl-Lewis X, sialyl-Lewis a, lacto-N-fucopentaose I,lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaoseV, LS-tetrasaccharide a, LS-tetrasaccharide b, LS-tetrasaccharide α,(3′) sialyllacto-N-neotetraose and derivatives thereof, serine,threonine, asparagine and peptides containing these amino acids andderivatives thereof, ceramide and derivaties thereof, and the like. Thecomplex carbohydrate precursor can be used at a concentration of from0.01 mM to 2.0 M.

Examples of the complex carbohydrate of the present invention includecomplex carbohydrates containing at least one sugar selected fromglucose, galactose, N-acetylglucosamine, N-acetylgalactosamine,glucuronic acid, mannose, N-acetylamannosamine, fucose, sialic acid,lactose, N-acetyllactosamine, lacto-N-biose, GlcNAcβ1-3Galβ1-4Glc,GlcNAcβ1-4Galβ1-4Glc, globotriose, Galα1-4Galβ1-4GlcNAc,2′-fucosyllactose, 3-fucosyllactose, 3′-sialyllactose, 6′-sialyllactose,3′-sialyl-N-acetyllactosamine, 6′-sialyl-N-acetyllactosamine,sialyllacto-N-biose, Lewis X, Lewis a, lacto-N-tetraose,lacto-N-neotetraose, lactodifucotetraose, 3′-sialyl-3-fucosyllactose,sialyl-Lewis X, sialyl-Lewis a, lacto-N-fucopentaose I,lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaoseV, LS-tetrasaccharide a, LS-tetrasaccharide b, LS-tetrasaccharide c,(3′) sialyllacto-N-neotetraose, lacto-N-difucohexaose I,lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose,disialyllacto-N-tetraose and derivatives thereof; and complexcarbohydrates which contain the just described complex carbohydrates.Specifically, they include complex carbohydrates which contain a sugarhaving a bond selected from Galβ1-3Glc, Galβ1-4Glc, Galβ1-3GlcNAc,Galβ1-4GlcNAc, Galβ1-3Gal, Galβ1-4Gal, Galβ1-3GalNAc, Galβ1-4GalNAc,Galα1-3Glc, Galα1-4Glc, Galα1-3GlcNAc, Galα1-4GlcNAc, Galα1-3Gal,Galα1-4Gal, Galα1-3GalNAc, Galα1-4GalNAc, GlcNAcβ1-3Gal, GlcNAcβ1-4Gal,GlcNAcβ1-6Gal, GlcNAcβ1-3Glc, GlcNacβ1-4Glc, GlcNAcβ1-3GlcNac,GlcNacβ1-4GlcNAc, GlcNacβ1-6GalNAc, GlcNAcβ1-2Man, GlcNAcβ1-4Man,GlcNAcβ1-6Man, GalNAcβ1-3Gal, GalNAcβ1-4Gal, GalNAcβ1-4GlcNAc,GalNAcα1-3GalNAc, Manβ1-4GlcNAc, Manα1-6Man, Manα1-3Man, Manα1-2Man,GlcUAβ1-4GlcN, GlcUAβ1-3Gal, GlcUAβ1-3GlcNAc, GlcUAβ1-3GalNac,NeuAcα2-3Gal, NeuAcα2-6Gal, NeuAcα2-3GlcNAc, NeuAcα2-6GlcNAc,NeuAcα2-3GalNAc, NeuAcα2-6GalNAc, NeuAcα2-8NeuAc, Fucα1-3Glc,Fucα1-4Glc, Fucα1-3GlcNAc, Fucα1-4GlcNAc, Fucα1-2Gal and Fucα1-6GlcNAc;and complex carbohydrates which contain the just described complexcarbohydrates. In this case, the number of sugars contained in thecomplex carbohydrate having the sugars may be 10 or below, or 6or below.

As specific processes for producing the complex carbohydrates,

(1) lactose can be formed from orotic acid, galactose and glucose bycarrying out an enzyme reaction using culture broths of a microorganismhaving the ability to express Neisseria-derivedβ1,4-galactosyltransferase (WO 96/10086), a microorganism having theability to produce UTP from a precursor of UTP and a microorganismhaving the ability to produce UDP-Gal from a sugar and UTP, or treatedproducts of these culture broths, as enzyme sources,

(2) N-acetyllactosamine can be formed from orotic acid, galactose andN-acetylglucosamine by carrying out an enzyme reaction using culturebroths of a microorganism having the ability to expressNeisseria-derived β1,4-galactosyltransferase (WO 96/10086), amicroorganism having the ability to produce UTP from a precursor of UTPand a microorganism having the ability to produce UDP-Gal from a sugarand UTP, or treated products of these culture broths, as enzyme sources,

(3) 3′-sialyllactose can be formed from orotic acid,N-acetylamannosamine, pyruvic acide and lactose by carrying out anenzyme reaction using culture broths of a microorganism having theability to express Neisseria-derived α2,3-sialytransferase (J. Biol,Chem., 271, 28271 (1996)), a microorganism having the ability to produceCTP from a precursor of CTP and a microorganism having the ability toproduce CMP-NeuAc from a sugar and CTP, or treated products of theseculture broths, as enzyme sources,

(4) 3′-sialyl-N-acetyllactosamine can be formed from orotic acid,N-acetylamannosamine, pyruvic acid and N-acetyllactosamine by carryingout an enzyme reaction using culture broths of a microorganism havingthe ability to express Neisseria-derived α2,3-sialyltransferase (J.Biol. Chem., 271, 28271 (1996)), a microorganism having the ability toproduce CTP from a precursor of CTP and a microorganism having theability to produce CMP-NeuAc from a sugar and CTP, or treated productsof these culture broths, as enzyme sources,

(5) 6′-sialyl-N-acetyllactosamine can be formed from orotic acid,N-acetylmannosamine, pyruvic acid and N-acetyllactosamine by carryingout an enzyme reaction using culture broths or COS-7 cell line havingthe ability to express chicken-derived α2,6-sialyltransferase (Eur. J.Biochem., 219, 375 (1994)), a microorganism having the ability toproduce CTP from a precursor of CTP and a microorganism having theability to produce CMP-NeuAc from a sugar and CTP, or treated productsof these culture broths, as enzyme sources,

(6) GlcNAcβ1-3Galβ1-4Glc can be formed from orotic acid,N-acetylglucosamine and lactose by carrying out an enzyme reaction usingculture broths of a microorganism having the ability to expressNeisseria-derived β1,3-N-acetylglucosaminyltransferase (WO 96/10086), amicroorganism having the ability to produce UTP from a precursor of UTPand a microorganism having the ability to produce UDP-GlcNAc from asugar and UTP, or treats products of these culture broths, as enzymesources,

(7) lacto-N-tetraose can be formed from orotic acid, galactose andGlcNAcβ1-3Galβ1-4Glc by carrying out an enzyme reaction using culturebroths of human melanoma cell line WM266-4 having the ability to produceβ1,3-galactosyltransferase (ATCC CRL 1676) or a tranformant such as ofnamalwa cell line KJM-1 having the ability to expressβ1,3-galactosyltransferase gene derived from human melanoma cell lineWM266-4 (Japanese Published Unexamined Patent Application No.181759/94), a microorganism having the ability to produce UTP from aprecursor of UTP and a microorganism having the ability to produceUDP-Gal from a sugar and UTP, or treats products of these culturebroths, as enzyme sources,

(8) lacto-N-neotetraose can be formed from orotic acid, galactose andGlcNAcβ1-3Galβ1-4Glc by carrying out an enzyme reaction using culturebroths of Escherichia coli (EMBO J., 9, 3171 (1990)) or Saccharomycescerevisiae (Biochem. Biophys. Res. Commun., 201, 160 (1994)) having theability to express β1,4-galactosyltransferase gene derived from humanHeLa cell line, a microorganism having the ability to produce UTP from aprecursor of UTP and a microorganism having the ability to produceUDP-Gal from a sugar and UTP, or treated products of these culturebroths, as enzyme sources,

(9) lacto-N-neotetraose can be formed from orotic acid, galactose andGlcNAcβ1-3Galβ1-4Glc by carrying out an enzyme reaction using culturebroths of a microorganism having the ability to expressNeisseria-derived β1,4-galactosyltransferase (WO 96/10086), amicroorganism having the ability to produce UTP from a precursor of UTPand a microorganism having the ability to produce UDP-GAl from a sugarand UTP, or treated products of these culture broths, as enzyme sources,

(10) lacto-N-neotetraose can be formed from orotic acid,N-acetylglucosamine, galactose and lactose by carrying out an enzymereaction using culture broths of a microorganism which can expressNeisseria-derived β1,3-N-acetylglucosaminyltransferase (WO 96/10086), amicroorganism having the ability to express Neisseria-derivedβ1,4-galactosyltransferase (WO 96/10086), a microorganism having theability to produce UTP from a precursor of UTP, a microorganism havingthe ability to produce UDP-GlcNAc from a sugar and UTP and amicroorganism having the ability to produce UDP-Gal from a sugar andUTP, or treated products of these culture broths, as enzyme sources,

(11) (3′) sialyllacto-N-neotetraose can be formed from orotic acid,N-acetylamannosamine, pyruvic acid and lacto-N-neotetraose by carryingout an enzyme reaction using culture broths of a microorganism havingthe ability to express Neisseria-derived α2,3-sialyltransferase (J.Biol. Chem., 271, 28271 (1996)), a microorganism having the ability toproduce CTP from a precursor of CTP and a microorganism having theability to produce CMP-NeuAc from a sugar and CTP, or treated productsof these culture broths, as enzyme sources,

(12) lacto-N-fucopentaose III can be formed from GMP, mannose andlacto-N-neotetraose by carrying out an enzyme reaction using culturebroths of namalwa cell line having the ability to express human-derivedα1,3-fucosyltransferase (J. Biol. Chem., 269, 14730 (1994)), amicroorganism having the ability to produce GTP from a precursor of GTPand a microorganism having the ability to produce GDC-Fuc from a sugarand GTP, or treated products these culture broths, as enzyme sources,

(13) lacto-N-fucopentaose III can be formed from GMP, mannose andlacto-N-neotetraose by carrying out an enzyme reaction using culturebroths of a microorganism having the ability to express Helicobacterpylori-derived α1,3-fucosyltransferase (J. Biol. Chem., 272, 21349 and21357 (1997)), a microorganism having the ability to produce GTP from aprecursor of GTP and a microorganism having the ability to produceGDP-Fuc from a sugar and GTP, or treated products of these culturebroths, as enzyme sources,

(14) globotriose can be formed from orotic acid, galactose and lactoseby carrying out an enzyme reaction using culture broths or amicroorganism having the ability to express Neisseria-derivedα1,4-galactosyltransferase (WO 96/10086), a microorganism having theability to produce UTP from a precursor of UTP and a microorganismhaving the ability to produce UDP-Gal from a sugar and UTP, or treatedproducts of these culture broths, as enzyme sources,

(15) globotriose can be formed from orotic acid, galactose and glucoseby carrying out an enzyme reaction using culture broths of amicroorganism having the ability to express Neisseria-derivedβ1,4-galactosyltransferase (WO 96/10086), a microorganism having theability to express Neisseria-derived α1,4-galactosyltransferase (WO96/10086), a microorganism having the ability to produce UTP from aprecursor of UTP and a microorganism having the ability to produceUDP-Gal from a sugar and UTP, or treated products of these culturebroths, as enzyme sources,

(16) Galα1-4Galβ1-4GlcNAc can be formed from orotic acid, galactose andN-acetyllactosamine by carrying out an enzyme reaction using culturebroths of a microorganism having the ability to expressNeisseria-derived α1,4-galactosyltransferase (WO 96/10086), amicroorganism having the ability to produce UTP from a precursor of UTPand a microorganism having the ability to produce UDP-Gal from a sugarand UTP, or treated products of these culture broths, as enzymessources,

(17) lacto-N-biose can be formed from orotic acid, galactose andN-acetylglucosamine by carrying out an enzyme reaction using culturebroths of an animal cell line having the ability to expresshuman-derived β1,3-galactosyltransferase (Japanese Published UnexaminedPatent Application No. 181759/94), a microorganism having the ability toproduce UTP from a precursor of UTP and a microorganism having theability to produce UDP-Gal from a sugar and UTP, or treated products ofthese culture broths, as enzyme sources,

(18) sialyllacto-N-biose can be formed from orotic acid,N-acetylamannosamine, pyruvic acid and lacto-N-biose by carrying out anenzyme reaction using culture broths of a microorganism having theability to express Neisseria-derived α2,3-sialyltransferase (J. Biol.Chem., 271, 28271 (1996)), a microorganism having the ability to produceCTP from a precursor of CTP and a microorganism having the ability toprodcue CMP-NeuAc from a sugar and CTP, or treated products of theseculture broths, as enzyme sources,

(19) sialyl-Lewis X can be formed from GMP, mannose and3′-sialyl-N-acetyllactosamine by carrying out an enzyme reaction usingculture broths of an animal cell line having the ability to expresshuman-derived α1,3-fucosyltransferase (J. Biol. Chem., 269, 14730(1994)), a microorganism having the ability to produce GTP from aprecursor of GTP and a microorganism having the ability to produceGDP-Fuc from a sugar and GTP, or treated products of these culturebroths, as enzyme sources,

(20) sialyl-Lewis a can be formed from GMP, mannose andsialyllacto-N-biose by carrying out an enzyme reaction using humanα1,3/1,4-fucosyltransferase (Carbohydrate Research, 190, 1 (1989)) andculture broths of a microorganism having the ability to produce GTP froma precursor of GTP and a microorganism having the ability to produceGDP-Fuc from a sugar and GTP, or treated products of these culturebroths, as enzyme sources, and

(21) Manα1-2Man can be formed from GMP and mannose by carrying out anenzyme reaction using culture broths of Escherichia coli having theability to express yeast-derived α1,2-mannosyltransferase (J. Org.Chem., 58, 3985 (1993)), a microorganism having the ability to produceGTP from a precursor of GTP and a microorganism having the ability toproduce GDP-Man from a sugar and GTP, or treated products of theseculture broths, as enzyme sources.

Processes for producing complex carbohydrates are not limited to theabove-mentioned examples, and any other sugar chain can be producedindustrially using a nucleotide precursor, a sugar and a complexcarbohydrate precursor as the sole starting materials, within the rangeof glycosyltransferases which can be combined with the sugar nucleotideproduction process described herein and of the substrate specificityacceptable by the enzymes.

Examples of the complex carbohydrate to be produced by the productionprocess of the present invention include

(1) complex carbohydrates involved in the infection with pathogenicmicroorganisms and viruses, such as complex carbohydrates which arerecognized as receptors for pathogenic microorganisms and viruses,

(2) complex carbohydrates which are recognized as receptors of toxinsproduced by pathogenic microorganisms and viruses,

(3) complex carbohydrates which are concerned, for example, in celladhesion, recognition of foreign substances and binding of various typesof lymphokine in the living body, and complex carbohydrates whichcontain one or a plurality of sugars such as glucose, galactose,N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, mannose,N-acetylmannosamine, fucose, sialic acid and the like, in a chemicallyacceptable binding mode. More specific examples include

(1) complex carbohydrates which are contained in the milk of human andanimals, and involved in the protection of infants from microbialinfection, for example, complex carbohydrates, such as lacto-N-tetraose,lacto-N-neotetraose and the like,

(2) receptor complex carbohydrates which recognize microorganisms, suchas Escherichia coli, Propionibacterium granulosium, Mycobacteriumtuberculosis, Moraxella cataxahlis, Candida albicans, Staphylococcussaprophyticus, Streptococcus pneumoniae, Streptococcus agalactiae,Pseudomonas aeruginosa, Actinomyces naeslundii, Neisseria gonorrhoeae,Helicobacter pylori, Haemophilus influenzae, and the like,

(3) receptor complex carbohydrates of viruses, such as influenza virus,coronavirus, sendai virus, newcastle disease virus, reovirus, rotavirus,AIDS (HIV) virus, and the like,

(4) receptor complex carbohydrates of protozoa, such as Cryptosporidium,Trypanosoma, and the like,

(5) receptor complex carbohydrates having the affinity for toxins, suchas cholera toxin, Escherichia coli heat-labile toxin, botulinum toxin,clostridial δ toxin, clostridial A toxin, Shiga toxin, Vero toxin, Shigatoxin-like toxin, Vibrio parahaemolyticus heat-stable toxin, tetanustoxin, and the like,

(6) cancer-related complex carbohydrates such as gangliosides (forexample, GD3, GM3, etc.), globoside glycolipids, and the like,

(7) complex carbohydrates which are concerned in the adhesion ofleukocytes to inflammatory regions and modification of their functions,such as sialyl-Lewis X sugar chain, and the like,

(8) complex carbohydrates concerned in autoimmune diseases, such asrheumatoid arthritis, IgA glomerulonephritis, and the like, and

(9) complex carbohydrates which are recognized by various lectin-likesubstances concerned in the recognition of foreign bodas and cancercells.

Determination of the complex carbohydrate formed in the aqueous mediumcan be carried out in accordance with known methods (Proc. Natl. Acad.Sci. USA., 85, 3289 (1988), Anal. Biochem., 174, 459 (1988)).

Recovery of the complex carbohydrate formed in the reaction solution canbe carried out in the usual way using activated carbon, an ion exchangeresin and the like, for example, N-acetyllactosamine can be recovered inaccordance with the process described in J. Org. Chem., 47, 5416 (1982).

Examples of the present invention are given below by way of illustrationand not by way of limitation.

BEST MODE OF CARRYING OUT THE INVENTION EXAMPLE 1 Construction ofRecombinant Plasmid Capable of Expressing galU and ppa

Construction process of recombinant plasmid pNT12 capable of expressinggalU and ppa is described in the following (FIGS. 1 and 2).

1) Construction of expression vector containing P_(L) promoter

Construction of pPA31 and pPAC31 as P_(L) promoter-containing expressionvectors carried out in the following manner (FIG. 1).

Escherichia coli JM109/pTrS30 (FERM BF-5407) which has tryptophanpromoter-containing plasmid pTrS30 and another Escherichia coli whichhas P_(L) promoter-containing plasmid pPA1 (Japanese PublishedUnexamined Patent Application No. 233798/88) and P_(L) promoter—andcI857 repressor-containing plasmid pPAC1 (FERMn BP-6054) were separatelyinoculated into LB medium (10 g/l Bacto-Tryptone (manufactured byDifco),5 g/l Yeast Extract (manufactured by Difco) and 5 g/l NaCl,adjusted to pH 7.2) and cultured at 30° C. for 18 hours.

From the cells obtained by the culturing, pTrS30, pPA1 and pPAC1 plasmidDNAs were isolated and purified by the above-mentioned known processes.

A 0.2 μg portion of the thus purified pTrS30 DNA was cleaved withrestriction enzymes PstI and ClaI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and than a fragment of 3.4 kbwas recovered using Gene Clean II Kit (manufactured by Bio101) . A 0.5μg portion of the purified pPA1 DNA was cleaved with restriction enzymesPstI and ClaI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 1.0 kb was recovered in the samemanner.

Using a ligation kit (TAKARA Ligation Kit, manufactured by Takara ShuzoCo., Ltd.), the fragments of 3.4 kb and 1.0 kb were subjected toligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 37° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, the P_(L)promoter-effected expression vector pPA31 was obtained. Structure of theplasmid was recognized by restriction enzyme cleavage (FIG. 1).

A 0.2 μg portion of the purified pPA31 DNA was cleaved with restrictionenzymes PstI and ClaI, the resulting DNA fragments were separated byagarose gel electrophoresis and then a fragment of 3.4 kb was recoveredusing Gene Clean II Kit. A 0.5 μg portion of the purified pPAC1 DNA wascleaved with restriction enzymes PstI and ClaI, the resulting DNAfragments were seperated by agarose gel electrophoresis and then afragment of 2.3 kb was recovered in the same manner.

Using a ligation kit, the fragmnts of 3.4 kb and 2.3 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 37° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, the P_(L)promoter-effected cI857 repressor-containing expression vector pPAC31was obtained. Structure of the plasmid was recognized by restrictionenzyme cleavage (FIG. 1).

2) Construction of galU expression plasmid

Chromosomal DNA of Escherichia coli W3100 was isolated and purified by aknown process (for example, Current Protocols in Molecular Biology, JohnWiley and Sons Inc. (1994)).

The sense strand DNA primer shown in SEQ ID NO: 1 and the antisencestrand DNA primer shown in SEQ ID NO: 2 were synthesized using 380A DNASynthesizer manufactured by Applied Biosystems.

The PCR process was carried out using the synthesized DNA strands asprimers, and the chromosomal DNA of the strain W3110 as the template.The PCR was affected using 40 μl of a reaction solution containing 0.04μg of the W3110 chromosomal DNA, 0.5 μM of each primer, 1.0 unit ofTAKARA Ex Taq (manufactured by Takara Shuzo Co., Ltd.), 4 μl of 10× ExTaq buffer (manufactured by Takara Shuzo Co., Ltd.) and 200 μM of eachdeoxyNTP, and repeating 30 cycles of the reaction, each cycle containing94° C. for 1 minute, 42° C. for 2 minutes and 72° C. for 3 minutes.

A {fraction (1/10)} volume of the reaction solution was subjected toagarose gel electrophoresis to verify amplification of the fragment ofinterest, and then the remaining reaction solution was mixed with thesame volume of phenol/chloroform (1 vol/1 vol) solution with saturatedTE (10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA). The mixture solutionwas centrifuged, and the thus obtained upper layer was mixed with 2volumes of cold ethanol and allowed to stand for 30 minutes at −80° C.The solution after standing was centrifuged to obtain a precipitate ofDNA. The precipitate was washed with 70% cold ethanol and dried in vacuoto recover the precipitate. Hereinafter, the steps starting from theaddition of phenol/chloroform solution with saturated TE until therecovery of the ethanol-washed DNA are referred to as an ethanolprecipitation process.

The DNA precipitate was dissolved in 20 μl of TE. Using 5 μl portion ofthe solution, DNA was cleaved with restriction enzymes HindIII andBamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 0.9 kb was recovered using GeneClean II Kit. A 0.2 μg portion of pPA31 DNA obtained in Example 1—1) wascleaved with restriction enzymes HindIII and BamHI, the resulting DNAfragments were separated by agarose gel electrophoresis and then afragment of 4.2 kb was recovered in the same manner.

Using a ligation kit, the fragments of 0.9 kb and 4.2 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli KY8415 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, galU expressionplasmid pNT9 was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 2).

3) Construction of simultaneous galU, ppa expression plasmid The sensestrand DNA primer shown in SEQ ID NO:3 and the antisence strand DNAprimer shown in SEQ ID NO:4 were synthesized, and the PCR was carriedout using the synthesized DNA strands as primers, and the chromosomalDNA of the strain W3110 as the template, under the same conditions asdescribed in the foregoing.

After completion of the PCR treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μg of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes BAMHI and SalI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 1.0kb was recovered using Gene Clean II Kit. A 0.2 μg portion of pNT9 DNAobtained in example 1-2) was cleaved with restriction enzyme BamHI andSalI, the resulting DNA fragments were separated by agarose galelectrophoresis and then a fragment of 4.9 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.0 kb and 4.9 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli KY8415 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, the galU, ppasimultaneous expression plasmid pNT12 was obtained. Structure of theplasmid was recognized by restriction enzyme cleavage (FIG. 2).

A 0.5 μg portion of the pNT12 DNA was cleaved with restriction enzymesEcoRI and SalI, the resulting DNA fragments were separated by agarosegel electrophoresis and then a fragment of 2.2 kb was recovered usingGene Clean II Kit. Separately from this, a 0.2 μg portion of pSTV28 DNA(manufactured by Takara Shuzo Co., Ltd.) was cleaved with restrictionenzymes EcoRI and SalI, the resulting DNA fragments were separated byagarose gel electrophoresis and then a fragment of 3.0 kb was recoveredin the same manner.

Using a ligation kit, the fragments of 2.2 kb and 3.0 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in the usual way, and the resulting transformant was spreadon LB agar medium containing 10 μg/ml chlorampenicol and then culturedovernight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin the usual way, the galU, ppa simultaneous expression plasmid pNT32was obtained. Structure of the plasmid was recognized by restrictionenzyme cleavage (FIG. 2).

EXAMPLE 2 Production of UDP-Glc

Escherichia coli KY8415/pNT12 obtained in Example 1 was inoculated intoa 1 L baffled conical flask containing 125 ml of LB medium supplementedwith 50 μg/ml ampicillin and cultured at 30° C. for 17 hours undershaking at 220 rpm. A 125 ml portion of the culture broth was inoculatedinto a 5 L culture vessel containing 2.5 L of an aqueous medium (withoutadjusting pH) which contains 10 g/l glucose, 12 g/l Bacto-Tryptone(manufactured by Difco), 24 g/l Yeast Extract (manufactured by Difco),2.3 g/l KH₂PO₄ (separate sterilization), 12.5 g/l K₂HPO₄ (separatesterilization) and 50 μg/ml ampicillin and cultured at 30° C. for 4hours and then at 40° C. for 3 hours, under conditions of 600 rpm and2.5 L/min of aeration.

During the culturing, pH of the medium was maintained at 7.0 using 28%aqueous ammonia. In addition, glucose was added during the culturingwhen required. The culture broth was centrifuged to obtain wet cells. Asoccasion demands, these wet cells can be preserved at −20° C. andutilized by thawing the cells prior to use.

Corynebacterium ammoniagenes ATCC 21170 was inoculated into a 300ml-baffled conical flask containing 20 ml of an aqueous medium of 50 g/lglucose, 10 g/l Polypeptone (manufactured by Nippon Seiyaku), 10 g/lYeast Extract (manufactured by Oriental Yeast), 5 g/l urea, 5 g/l(NH₄)₂SO₄, 1 g/l KH₂PO₄, 3 g/l K₂HPO₄, 1 g/l MgSO₄.7H₂O, 0.1 g/l CaCl₂.10 mg/l FeSO₄.7H₂O, 10 mg/l ZnSO₄.7H₂O, 20 mg/l MnSO₄•4-6H₂O, 20 mg/lL-cysteine, 10 mg/l calcium D-pantothenate, 5 mg/l vitamin B₁, 5 mg/lnicotinic acid and 30 μg/l biotin (adjusted to pH 7.2 with 10 N NaOH)and cultured at 28° C. for 24 hours under shaking at 220 rpm.

A 20 ml portion of the culture broth was inoculated into a 2 L baffledconical flask containing 250 ml of the same aqueous medium and culturedat 28° C, for 24 hours under shaking at 220 rpm. The thus obtainedculture broth was used as a seed culture broth.

A 250 ml portion of the seed culture broth was inoculated into a 5 Lculture vessel containing 2.25 L of an aqueous medium of 150 g/lglucose, 5 g/l meat extract (manufactured by Kyokuto PharmaceuticalIndustry), 10 g/l KH₂PO₄, 10 g/l K₂HPO₄, 10 g/l MgSO₄.7H₂O, 1.0 g/lCaCl₂.2H₂O 20 mg/l FeSO₄.7H₂O, 10 mg/l ZnSO₄.7H₂O, 20 mg/l MnSO₄.4-6H₂O(separate sterilization), 15 mg/l β-alanine (separate sterilization), 20mg/l L-cystaine, 100 μg/l biotin, 2 g/l urea and 5 mg/l vitamin B₁(separate sterilization) (adjusted to pH 7.2 with 10 N NaOH) andcultured at 32° C. for 24 hours under conditions of 600 rpm and 2.5L/min of aeration. During the culturing, pH of the culture broth wasmaintained at 6.8 using 28% aqueous ammonia.

The culture broth was centrifuged to obtain wet cells. As occasiondemands, these wet cells can be prepared at −20° C. and utilized bythawing the cells prior to use.

A 30 ml portion of a reaction solution having a composition of 40 g/lEscherichia coli KY8415/pNT12 wet cells, 150 g/l Corynabacteriumammoniagenes ATCC 21170 wet cells, 100 g/l glucose, 20 g/l KH₂PO₄, 5 g/lmgSO₄.7H₂O, 5 g/l phytic acid, 21.2 g/l orotic acid (potassium salt), 4g/l Nymean S-215 and 10 ml/l xylene was put into a 200 ml beaker, and 21hours of the reaction was carried out at 32° C. under stirring thereaction solution with a magnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOh, and glucose and KH₂PO₄ were added when required.

By the reaction, 43.9 g/l UDP-Glc (2Na salt) was formed in the reactionsolution.

EXAMPLE 3

Construction of Recombinant Plasmid Capable of Expressing galT and galK

Construction process of recombinant plasmid pNT25 capable of expressinggalT and galK is described in the following (FIG. 3).

The sense strand DNA primer shown in SEQ ID NO:5 and the antisencestrand DNA primer shown in SEQ ID NO:6 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of the strain W3110 as the template, under the sameconditions as described in the following.

After completion of the PCR treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes HindIII and HincII, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 2.3kb was recovered using Gene Clean II Kit. A 0.2 μg portion ofpBluescript II SK+ DNA was cleaved with restriction enzymes HindIII andEcoRV, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 3.0 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 2.3 kb and 3.0 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and the cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, plasmid pNT19containing galT and galK genes was obtained. Structure of the plasmidwas recognized by restriction enzyme cleavage (FIG. 3).

A 0.5 μg portion of the pNT19 DNA was cleaved with restriction enzymesClaI and BamHI, the resulting DNA fragments were separated by agarosagel electrophoresis and then a fragment of 2.3 kb was recovered in thesame manner. A 0.2 μg portion of the pPAC31 DNA obtained in Example 1-1)was cleaved with restriction enzymes ClaI and BamHI, the resulting DNAfragments were separated by agarose gel electrophoresis and then afragment of 5.5 kb was recovered in the same manner.

Using a ligation kit, the fragments of 2.3 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM52 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, plasmid pNT25capable of expressing galT and galK simultaneously was obtained.Structure of the plasmid was recognized by restriction enzyme cleavage(FIG. 3).

EXAMPLE 4 Production of UDP-Gal

1) Preparing of galT, galK, galU, ppa expression strain

Using the pNT32 DNA obtained in Examples 1-3), Escherichia coliNM522/pNT25 was transformed in accordance with the above-mentioned knownprocess, and the resulting transformant was spread on LB agar mediumcontaining 50 μg/ml ampicillin and 10 μg/ml chloramphanicol and thencultured overnight at 30° C. By selecting the thus grown transformants,Escherichia coli NM522/pNT25/pNT32 was obtained as the galT, galK, galU,ppa expression strain.

2) Production of UDP-Gal

Escherichia coli NM522/pNT25/pNT 32 obtained in Example 4-1) wascultured in the same manner as in Example 2, and the culture broth wascentrifuged to obtain wet cells. Also, Corynabacterium ammoniagenes ATCC21170 was cultured in the same manner as in Example 2, and the culturebroth was centrifuged to obtain wet cells, As occasion demands, the wetcells can be prepared at −20° C. and utilized by thawing them prior touse.

A 2 L portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25/pNT32 wet cells, 150 g/l Corynabacteriumammoniagenes ATCC 21170 wet cells, 80 g/l glucose, 20 g/l galactose, 15g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 21.2 g/l orotic acid(potassium salt), 4 g/l Nymean S-215 and 10 ml/l xylene was put into 5 Lculture vessel, and 26 hours of the reaction was carried out at 32° C.under stirring the reaction solution at 600 rpm with an aeration rate of1 L/min.

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and glucose, galactose and KH₂PO₄ were added whenrequired.

By the reaction, 47.4 g/l UDP-Gal (2Na salt) ws formed in the reactionsolution.

EXAMPLE 5 Construction of Recombinant Plasmid Capable of Expressing galTand galK in Corynabacterium ammoniagenes

Construction process of recombinant plasmid pTK7 capable of expressingEscherichia coli-derived galT and galK in Corynabacteriuim ammoniagenesis described in the following (FIG. 4).

1) Construction of pCG116

Plasmid pCG116 capable of replicating in Corynabacterium ammoniagenesewas constructed in the following manner.

A 0.5 μg portion of plasmid pCG11 (Japanese Published Examined PatentApplication No. 91827/94) DNA was cleaved with restriction enzymes PstIand StuI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 6.5 kb was recovered using GeneClean II Kit.

On the other hand, a 1.0 μg portion of plasmid pUC19 DNA was cleavedwith a restriction enzyme EcoRI and then blunt-ended using DNA BluntingKit (manufactured by Takara Shuzo Co., Ltd.). The DNA thus blunt-endedwas cleaved with a restriction enzyme PstI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 43bp was recovered using MERmaid Kit (manufactured by Bio101).

Using a ligation kit, the fragments of 6.5 kb and 43 bp were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Corynabacterium ammoniagenes ATCC21170 was transformed by the electroporation method (FEMS Microbiol.Lett., 65, 299 (1989), and the resulting transformant was spread on LBagar medium containing 100 μg/ml spectinomycin and then cultured at 30°C. for 2 days.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the known process (J. Bacteriol., 159, 306 (1984)),plasmid pCG116 was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 4).

2) Construction of pTK7 capable of expressing galT and galK

A 1.0 μg portion of the galT and galK expression plasmid pNT25 DNAobtained in Example 3 was cleaved with restriction enzymes XhoI andBamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 3.5 kb was recovered using GeneClean II Kit.

On the other hand, a 0.5 μg portion of the plasmid pCG116 DNA preparedin Example 5-1) was cleaved with a restriction enzymes SalI and BamHI,the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 6.5 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 3.5 kb and 6.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Corynabacterium ammoniagenes ATCC21170 was transformed by the electroporation process, and the resultingtransformant was spread on LB agar medium containing 100 μg/mlspectinomycin and then cultured at 30° C. for 2 days.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the known process, plasmid pTK7 capable of expressinggalT and galK simultaneously was obtained. Structure of the plasmid wasrecognized by restriction enzyme cleavage (FIG. 4).

EXAMPLE 6 Production of UDP-Gal

Cornabacterium ammoniagenes ATCC 21170/pTK7 obtained in Example 5 wascultured by the same process as described in Example 2 at 32° C. for 20hours and then for 4 hours, and the thus obtained culture broth wascentrifuged to obtain wet cells. As occasion demands, the wet cells canbe preserved at −20° C. and utilized by thawing them prior to use.

A 30 ml portion of a reaction solution having a composition of 150 g/lCorynabacterium ammoniagenes ATCC 21170/pTK7 wet cells, 40 g/l fructose,20 g/l galactose, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid,10.6 g/l orotic acid (potassium salt), 4 g/l Nymean S-215 and 10 ml/lxylene was put into a 200 ml beaker, and 22 hours of the reaction wascarried out at 32° C. under stirring the reaction solution with amagnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and fructose, galactose and KH₂PO₄ were added whenrequired.

By the reaction, 7.2 g/l UDP-Gal (2Na salt) was formed in the reactionsolution.

EXAMPLE 7 Construction of glmU, ppa, pgm, glmM, glk and pfkB ExpressionPlasmid

1) Construction of glmU and ppa expression plasmid

The sense strand DNA primer shown in SEQ ID NO:7 and the antisencestrand DNA primer shown in SEQ ID NO:8 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of the strain W3110 as the template, under the sameconditions as described in the foregoing.

After completion of the PCR treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The DNA precipitate was dissolvedin 20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes HindIII and BamHI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 1.4kb was recovered using Gene Clean II Kit. A 0.5 μg portion of pPA31 DNAobtained in Example 1-1) was cleaved with restriction enzymes HindIIIand BamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 4.2 kb was recovered in the samemanner.

Using a ligation kit, the fragment of 1.4 kb and 4.2 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli KY8415 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, glmU expressionplasmid pNT10 was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 5).

A 0.5 μg portion of the pNT12 DNA obtained in Example 1-3) was cleavedwith restriction enzymes BamHI and SalI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 1.0kb was recovered in the same manner. A 0.2 μg portion of the justdescribed pNT10 DNA was cleaved with restriction enzymes BamHI and SalI,the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 5.3 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.0 kb and 5.3 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli KY8415 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, the glmU, ppasimultaneous expression plasmid pNT14 was obtained. Structure of theplasmid was recognized by restriction enzyme cleavage (FIG. 5).

2) Construction of pgm expression plasmid

The sense strand DNA primer shown in SEQ ID NO:9 and the antisencestrand DNA primer shown in SEQ ID NO:10 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of the strain W3110 as the template, under the sameconditions as described in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by thoethanol precipitation process. The precipitate was dissolved in 20 μlTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes ClaI and BamHI, the resulting DNA fragment wereseparated by agarose gel electrophoresis and then a fragment of 1.8 kbwas recovered using Gene Clean II Kit. A 0.2 μg portion of the pPAC31DNA obtained in Example 1-1) was cleaved with restriction enzymes ClaIand BamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 5.5 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.8 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with th above-mentioned known process, pgm expressionplasmid pNT24 was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 6).

3) Construction of glmM expression plasmid

The sense strand DNA primer shown in SEQ ID NO:11 and the antisencestrand DNA primer shown in SEQ ID NO:12 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Escherichia coli W3110 as the template, under thesame conditions as described in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by thethanol precipitation process. The precipitate was dissolved in 20 μl ofTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes ClaI and BamHI, the resulting fragments wereseparated by agarose gal electrophoresis and then a fragment of 1.6 kbwas recovered using Gene Clean II Kit. A 0.2 μg portion of the pAC31 DNAobtained in Example 1-1) was cleaved with restriction enzymes ClaI andBamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 5.5 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.6 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above-mentioned known process, andthe resulting transformant was spread on LB agar medium containing 50μg/ml ampicillin and then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the above-mentioned known process, glmM expressionplasmid pNT44 was obtained. Structure of the plasmid was recognized byrestriction enzymes cleavage (FIG. 7).

4) Construction of glk expression plasmid

The sense strand DNA primer shown in SEQ ID NO:13 and the antisencestrand DNA primer shown in SEQ ID NO:14 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Escherichia coli W3110 as the plate, under the sameconditions as described in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by theethanol precipitation process. The precipitate was dissolved in 20 μl ofTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes HindIII and BamHI, the resulting DNA fragments wereseparated by agarose gal electrophoresis and then a fragment of 0.5 kbwas recovered using Gene Clean II Kit.

A 0.2 μg portion of the pPA31 DNA obtained in Example 1-1) was cleavedwith restriction enzymes HindIII and BamHI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 4.2kb was recovered in the same manner.

Using a ligation kit, the fragments of 0.5 kb and 4.2 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NW522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin the usual way, plasmid pNT45 containing a part of glk was obtained.Structure of the plasmid was recognized by restriction enzyme cleavage(FIG. 8).

The PCR was carried out under the same conditions described above, theDNA contained in 5 μl portion of the thus obtained DMA solution wascleaved with a restriction enzyme HindIII, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragments of0.5 kb was recovered in the same manner. A 0.2 μg portion of the pNT45DNA obtained by the just described process was cleaved with therestriction enzyme HindIII and subjected to a dephosphorylationtreatment with alkaline phosphatase, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragment of 4.7 kbwas recovered in the same manner.

Using a ligation kit, the fragments of 0.5 kb and 4.7 kb subjected toligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual way, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual way, glk expression plasmid pNT46 wasobtained. Structure of the plasmid was recognized by restriction enzymecleavage (FIG. 8).

5) Construction of pfkB expression plasmid

The sense strand DNA primer shown in SEQ ID NO:15 and the antisencestrand DNA primer shown in SEQ ID NO:16 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of the strain W3110 as the template, under the sameconditions as described in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by theethanol precipitation process. The precipitate was dissolved in 20 μl ofTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes HindIII and EcoRV, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragment of 1.3 kbwas recorded using Gene Clean II Kit. A 0.2 μg portion of pBluescript IISK+ DNA was cleaved with restriction enzymes HindIII and EcoRV, theresulting DNA fragments were separated by agarose gel electrophoresisand then a fragment of 3.0 kb was recovered in the same manner.

Using a ligation kit, the fragments of 1.3 kb and 3.0 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plamid for the thus grown colonies of the transformantin accordance with the usual process, plasmid pNT43 containing the pfkBgene was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 9).

A 0.5 μg portion of the pNT43 DNA was cleaved with restriction enzymesClaI and SacI, the resulting DNA fragments seated by agarose galelectrophoresis and then a fragment of 1.3 kb was recovered in the samemanner.

A 0.2 μg portion of the pPAC31 DNA obtained in Example 1-1) was cleavedwith restriction enzymes ClaI and SacI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragments of 5.7 kbwas recovered in the same manner.

Using a ligation kit, the fragments of 1.3 kb and 5.7 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coil NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, pfkB expression plasmid pNT47 wasobtained. Structure of the plasmid was recognized by restriction enzymecleavage (FIG. 9).

EXAMPLE 8 Production of UDP-GlcHAc

Escherichia coli KY8415/pNT14,NM522/pNT24, NM522/pNT44 and NM522/pNT47obtained in Example 7 were cultured in the same manner as in Example 2,and each of the thus obtained culture broths were centrifuged to obtainwet cells. As occasion demands, these wet cells can be preserved at −20°C. and utilized by thawing the calls prior to use.

A 0.1 ml portion of a reaction solution having a composition of 6 g/lEscherichia coli NM522/pNT24 wet cells, 6 g/l NM522/pNT47 wet cells, 100mM Tris-HCl buffer (pH 8.0), 6 mM MgCl₂.6H₂O, 10 mM glucose-6-phosphate,2.5 mM fructose-6-phosphate, 2.5 mM ATP and 4 g/l Nymean S-215 was putinto a 1.5 ml tube, and 1 hour of the reaction was carried out at 37° C.The reaction solution was treated at 65° C. for 5 minutes and shortagein cells and substances was suspended until 0.3 g/l Escherichia coliKY8415/pNT14 wet cells, 6 g/l NM522/pNT44 wet cells, 5 mMglucosamine-6-phosphate, 5 mM acetyl-CoA and 5 mM UTP, and then 30minutes of the reaction was carried out at 37° C. to find that 2.5 mM(1.6 g/l) UDP-GlcNac (2Na salt) was formed in the reaction solution. Inthis connection, when Escherichia coli NM522/pNT24 wet cells orNM522/pNT47 wet cells were not added, formed amounts of the UDP-GlcNAcwere 0.08 mM and 0.16 mM, respectively.

These results indicate that Glc-1, 6-P2 necessary for the expression ofglmM activity can be provided by the combination of a pgm expressionstrain with a pfkB expression strain.

EXAMPLE 9 Production of UDP-GlcNAc

Escherichia coli KY8415/pNT14, NM522/pNT24, NM522/pNT44, NM522/pNT46 andNH522/pNT47 obtained in Example 7 were cultured in the same manner as inExample 2, and each of the thus obtained culture broths was centrifugedto obtain wet cells. Also, Corynebacterium ammoniagenes ATCC 21170 wascultured in the same manner as in Example 2, and the thus obtainedculture broth was centrifuged to obtain wet cells. As occasion demands,these wet cells can be preserved at −20° C. and utilized by thawing thecells prior to use.

A 30 ml portion of a reaction solution having a composition of 10 g/lwet cells of each of Escherichia coli KY8415/pNT14, NM522/pNT24,NM522/pNT44, NM522/pT47 and NM522/pNT46, 150 g/l Corynebacteriumammoniagenes ATCC 21170 wet cells, 50 g/l fructose, 80 g/l glucosaminehydrochloride, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/ phytic acid, 10 g/lorotic acid (potassium salt), 4 g/l Nymeen S-215 and 10 ml/l xylene wasput into a 200 ml beaker, and 10 hours of the reaction was carried outat 32° C. under stirring the reaction solution with a magnetic stirrer(900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and fructose and KH₂PO₄ were added when required.

By the reaction, 6.2 g/l UDP-GlcNAc (2Na salt) was formed in thereaction solution.

EXAMPLE 10 Construction of galK Expression Plasmid

A 0.5 μg portion of the pNT25 DNA obtained in Example 3-1) was cleavedwith restriction enzymes ClaI and EcoRV, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 6.7kb was recovered using Gene Clean II kit. The thus recovered DNA wasblunt-ended using DNA Blunting Kit and then subjected to ligationreaction for 16 hours at 16° C. using a ligation kit.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, galK expression plasmid pNT54 wasobtained. Structure of the plasmid was recognized by restriction enzymecleavage (FIG. 10).

EXAMPLE 11 Production of UDP-GlcNAc

Escherichia coli NM522/pNT54 obtained in Example 10 was cultured in thesame manner as in Example 2, and the culture broth was centrifuged toobtain wet cells. Also, Corynebacterium ammoniagenes ATCC 21170 wascultured in the same manner as in Example 2, and the culture broth wascentrifuged to obtain wet cells. As occasion demands, these wet cellscan be present at −20° C. and utilized by thawing the cells prior touse.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT54 wet cells, 150 g/l Corynebacteriumammoniagenes ATCC 21170 wet cells, 40 g/l fructose, 67 g/lN-acetylglucosamine, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid,10 g/l orotic acid (potassium salt), 4 g/l Nymeen S-215 and 10 ml/lxylene was put into a 200 ml, and 27 hours of the reaction was carriedout at 32° C. under stirring the reaction solution with a magneticstirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and fructose and KH₂PO₄ were added when required.

By the reaction, 17.1 g/l UDP-GlcNAc (2Na salt) was formed in thereaction solution.

EXAMPLE 12 Simultaneous Production of UDP-GlcNAc and UDP-Gal

The strain NM522/pNT25 obtained in Example 3 was cultured in the samemanner as in Example 2, and the culture broth was centrifuged to obtainwet calls. Also, Corynebacterium ammoniagenes ATCC 21170 was cultured inthe same manner as in Example 2, and the culture broth was centrifugedto obtain wet cells. As occasion demands, these wet cells can bepreserved at −20° C. and utilized by thawing the cells prior to use.

A 30 ml portion of a reaction solution having a composition of 25 g/lEscherichia coli NM522/pNT25 wet cells, 150 g/l Corynebacteriumammoniagenes ATCC 21170 wet cells, 60 g/l fructose, 50 g/lN-acetylglucosamine, 40 g/l galactose, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O,5 g/l phytic acid, 10 g/l orotic acid (potassium salt), 4 g/l NymeenS-215 and 10 ml/l xylene was put into a 200 ml beaker, and 24 hours ofthe reaction was carried out at 32° C. under stirring the reactionsolution with a magnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO₄ was added when required.

By the reaction, 11.4 g/l UDP-GlcNAc (2Na salt) and 18 g/l UDP-Gal (2Nasalt) formed in the reaction solution.

EXAMPLE 13 Construction of Plasmid Capable of Expressing manB, manC, pgman pfkB

1) Construction of manB, manC expression plasmid

The sense strand DNA primer shown in SEQ ID NO: 17 and the antisencestrand DNA primer shown in SEQ ID No: 18 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Escherichia coli W3110 as the template, under thesame conditions as described in the foregoing.

After completion of the PCR treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes HindIII and BamHI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 3.0kb was recovered using Gene Clean II Kit. A 0.2 μg portion ofpBluescript II SK+ DNA was cleaved with restriction enzymes HindIII andBamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 3.0 kb was recovered in the samemanner.

Using a ligation kit, both of the fragments of 3.0 kb were subjected toligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid for the thus grown colonies of the transformantin accordance with the above mentioned known process, plasmid pNK6containing manC and manB was obtained. Structure of the plamid wasrecognized by restriction enzyme cleavage (FIG. 11).

A 0.5 μg portion of the pNK6 DNA was cleaved with restriction enzymesClaI and BamHI, the resulting DNA fragments were separated by agarosegel electrophoresis and then a fragment of 3.0 kb was recovered. A 0.2μg portion of pPAC31 DNA obtained in Example 1-1) was cleaved withrestriction enzymes ClaI and BamHI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragment of 5.5 kbwas recovered.

Using a ligation kit, the fragments of 3.0 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, manC, manB expression plasmid pNK7was obtained. Structure of the plasmid was recognized by restrictionenzyme cleavage (FIG. 11).

2) Construction of pgm, pfkB simultaneous expression plasmid

A 0.5 μg portion of the pNT24 DNA obtained in Example 7 was cleaved withrestriction enzymes XhoI and BamHI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragment of 3.0 kbwas recovered using Gene Clean II Kit. On the other hand, a 0.2 μgportion of pSTV28 DNA (manufactured by Takara Shuzo Co. , Ltd.) wascleaved with restriction enzymes SalI and BamHI, the resulting DNAfragments were separated by agarose gel electrophoresis and then afragment of 3.0 kb was recovered in the same manner.

Using a ligation kit, both of the fragments of 3.0 kb were subjected toligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 10 μl/mlchloramphanicol and then cultured overnight at 30° C.

By extracting a plasmid fr the thus grown colonies of the transformantin accordance with the usual process, plasmid pNT53 containing the pgmgene was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 12).

The sense strand DNA primer shown in SEQ ID NO:19 was synthesized, andthe PCR was carried out using the sense strand DNA primer and theantisence strand DNA primer shown in SEQ ID NO:16, and the pNT47 DNAobtained in Example 7 as the template, under the same conditions asdescribed in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by theethanol precipitation process. The precipitate was dissolved in 20 μl ofTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes EcoRV and BglII, the resulting DNA fragments wereseparated by agarose gal electrophoresis and then a fragment of 1.3 kbwas recovered in the same manner. A 0.2 μg portion of pNT53 DNA wascleaved with restriction enzymes SmaI and BamHI, the resulting DNAfragments were separated by agarose gel electrophoresis and then afragment of 6.0 kb was recovered in the same manner.

Using a ligation kit, the fragments of 1.3 kb and 6.0 kb subjected toligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 10 μg/mlchloramphanicol and cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, plasmid pNT55 capable ofexpressing pgm and pfkB was obtained. Structure of the plasmid wasrecognized by restriction enzyme cleavage (FIG. 12).

EXAMPLE 14 Production of GDP-Man

1) Preparation of manB, manC, pgm, pfkB expression strain

Using the pNT55 DNA obtained in Example 13-2), Escherichia coliNM522/pnK7 was transformed in accordance with the usual process, and theresulting transformant was spread on LB agar medium containing 50 μg/mlampicillin and 10 μg/ml chloramphanicol and cultured overnight at 30° C.By selecting the thus grown transformants, Escherichia coliNM522/pNK7/pNT55 was obtained as a manB, manC, pgm, pfkB expressionstrain.

2) Production of GDP-Man

Escherichia coli NM522/pNK7/pNT55 obtained in the above step 1) andEscherichia coli NM522/pNT46 obtained in Example 7 were culturedseparately in the same manner as in Example 2, and each of thus obtainedthe culture broths was centrifuged to obtain wet cells. Also,Corynebacterium ammoniagenes ATCC 21170 was cultured in the same manneras in Example 2, and the culture broth was centrifuged to obtain wetcells. As occasion demands, these wet cells can be preserved at −20° C.and utilized by thawing the cells prior to use.

A 30 ml portion of a reaction solution having a composition of 25 g/lEscherichia coli NM522/pNK7/pNT55 wet cells, 25 g/l the NM522/pNT46 wetcells, 150 g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 60 g/lfructose, 50 g/l mannose, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phyticacid, 60 g/l GMP (2Na, 7H₂O salt), 4 g/l Nymeen S-215 and 10 ml/l xylenewas put into a 200 ml beaker, and 24 hours of the reaction was carriedout under stirring the reaction solution with a magnetic stirrer (900rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO₄ was added when required.

By the reaction, 14.6 g/l GDP-Man (2Na, 1H₂O salt) was formed in thereaction solution.

EXAMPLE 15 Construction of gmd, wcaG Expression Plasmid

The sense strand DNA primer shown in SEQ ID NO:20 and the antisencestrand DNA primer shown in SEQ ID NO:21 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Escherichia coli W3110 as the template, under thesame conditions as described in the foregoing.

After completion of the PCR, a precipitate of DNA was obtained by theethanol precipitation process. The precipitate was dissolved in 20 μl ofTE. Using 5 μl portion of the solution, the DNA was cleaved withrestriction enzymes HindIII and XhoI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a fragment of 2.3 kbwas recovered using Gene Clean II Kit.

A 0.2 μg portion of the pPA31 DNA obtained in Example 1-1) was cleavedwith restriction enzymes HindIII and SalI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 3.9kb was recovered in the same manner.

Using a ligation kit, the fragments of 2.3 kb and 3.9 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, plasmid pNK8 containing gmd andwcaG was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 13).

EXAMPLE 16 Production of GDP-Fuc

Escherichia coli NM522/pnK7/pNT55 obtained in Example 14, NM522/pNK8obtained in Example 15 and NM522/pNT46 obtained in Example 7 werecultured in the same manner as in Example 2, and each of the thusobtained culture broths was centrifuged to obtain wet cells. Also,Corynebacterium ammoniagenes ATCC 21170 was cultured in the same manneras in Example 2, and the culture broth was centrifuged to obtain wetcells. As occasion demands, these wet cells can be preserved at −20° C.and utilized by thawing the cells prior to use.

A 30 ml portion of a reaction solution having a composition of 25 g/lEscherichia coli NM522/pNK7/pNT55 wet cells, 25 g/l Escherichia coliNM522/pNK8 wet cells, 25 g/l Escherichia coli NM522/pNT46 wet cells, 150g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 40 g/l fructose,60 g/l mannose, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 60g/l GMP (2Na/7H₂O salt), 4 g/l Nymeen S-215 and 10 ml/l xylene was putinto a 200 ml beaker, and 24 hours of the reaction was carried out at32° C. under stirring the reaction solution with a magnetic stirrer (900rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO₄ was added when required.

By the reaction, 1.0 g/l GDP-Fuc (2.5Na, 1H₂O salt) was formed in thereaction solution.

EXAMPLE 17 Construction of neuA Expression Plasmid

Chromosomal DNA of Escherichia coli K235 (ATCC 13027) was prepared inthe same manner as in Example 1.

The sense strand DNA primer shown in SEQ ID NO:22 and the antisencestrand DNA primer shown in SEQ ID NO:23 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Escherichia coli K235 (ATCC 13027) as the template,under the same conditions as described in the foregoing.

After completion of the PCR treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes EcoRI and BamHI, the resulting DNA fragmentswere separated by agarose gal electrophoresis and then a fragment of 1.3kb was recovered using Gene Clean II Kit. A 0.2 μg portion ofpBluescript II SR+ DNA was cleaved with restriction enzymes EcoRI andRI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 3.0 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.3 kb and 3.0 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, plasmid pTA12 containing the neuAgene was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 14).

A 0.5 μg portion of the pTA12 M was cleaved with restriction enzymesClaI and BamHI, the resulting DNA fragments were separated by agarosegal electrophoresis and then a fragment of 1.3 kb was recovered in thesame manner. A 0.2 μg portion of the pPAC31 DNA obtained in Example 1-1)was cleaved with restriction enzymes ClaI and BamHI, the resulting DNAfragments were separated by agarose gel electrophoresis and then afragment of 5.5 kb was recovered in the same manner.

Using a ligation kit, the fragments of 1.3 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, neuA expression plasmid pTA14 wasobtained. Structure of the plasmid was recognized by restriction enzymecleavage (FIG. 14).

EXAMPLE 18 Production of CMP-NeuAc

Escherichia coli NM522/pTA14 obtained in Example 17, C600/pNAL1 (Appl.Environ. Microbiol., 51, 562 (1986)) and JF646/pMW5 (J. Biol. Chem.,261, 5568 (1986)) were separately cultured in the same manner as inExample 2, and each of the thus obtained culture broths was centrifugedto obtain wet cells. Also, Corynabacterium ammoniagenes ATCC 21170 wascultured in the same manner as in Example 2, and the culture broth wascentrifuged to obtain wet cells. An occasion demands, these wet cellscan be preserved at −20° C. and utilized by thawing the cells prior touse.

A 30 ml portion of a reaction solution having a position of 50 g/lEscherichia coli NM522/pTA14 wet cells, 15 g/l Escherichia coliC600/pNALl1 wet cells, 25 g/l Escherichia coli JF646/pMW5 wet cells, 150g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 10 g/l oroticacid (potassium salt), 20 g/l pyruvic acid (Na salt), 40 g/l fructose,10 g/l N-acetylmannosamine, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/lphytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylene was put into a 200 mlbeaker, and 24 hours of the reaction was carried out at 32° C. understirring the reaction solution with a magnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO₄ was added when required.

By the reaction, 2.7 g/l CMP-NeuAc (Na salt) was formed in the reactionsolution.

EXAMPLE 19 Production of lacto-N-tetraose

1) Preparation of β1,3-galactosyltransferase

Namalwa cell line KJM-1 transformed with plasmid pAMoERSAW1 (JapanesePublished Unexamined Patent Application No. 181759/94) containing a geneencoding a fusion protein of the IgG binding region of protein A withβ1,3-galactosyltransferase was suspended in 30 ml of RPMI1640.ITPSGFmedium containing 0.5 mg/ml G418 (manufactured by Gibco), to a densityof 5×10⁴ cells/ml, and cultured at 37° C. for 8 days in a CO₂ incubator.

Cells were removed from the culture broth by centrifugation, and thesupernatant was recovered. As occasion demands, the supernatant can bestored at −80° C. and utilized by thawing the supernatant prior to use.

To the culture supernatant in which the fusion protein of the IgGbinding region of protein A with β1,3-galactosyltransferase has beenformed were added sodium azide to a final concentration of 0.1% and then50 μl of IgG Sepharose (manufactured by Pharmacia) which has beenpre-treated in accordance with the manufacturer's instructions. Themixture was stirred overnight gently at 4° C.

After the stirring, the β1,3-galactosyltransferase-linked IgG Sepharosewas by centrifugation and washed three times with 1 ml of RFM1640.ITPSGFdrum, and then the IgC Sepharose was used as the only source ofβ1,3-galactosyltransferase.

2) Production of lacto-N-tetraose

Lacto-N-neotetraose (manufactured by Oxford Glycosystems) wasfluorescence-labeled by 2-aminopyridine in accordance with a knownprocess (Agric. Biol. .Chem, 54, 2169 (1990)) and mixed with 0.1 Uβ-galactosidase (manufactured by Seikagu Kogyo K.K.), and then themixture was allowed to react for 16 hours at 37° C. to remove thegalactose at the non-reducing end.

The reaction solution was heated at 100° C. for 5 minutes to inactivateβ-galactosidase.

GlcNAcβ1-3Galβ1-4Glc obtained by the reaction was used as a complexcarbohydrate precursor.

A 36 μl portion of a reaction solution containing 0.5 mM of the complexcarbohydrate precursor, 0.5 U the β1,3-galactosyltransferase linked IgGSepharose obtained in the above step 1), 6 μl of a reaction solutioncontaining UDP-Gal (5 mM) obtained in Example 4, 100 mM Tris-HCl (pH7.9), 10 mM MnCl₂ and 2 mM β-mercaptoethanol was allowed to stand for 65hours at 32° C. to effect the reaction.

After completion of the reaction, amount of the product accumulated inthe reaction solution was measured by HPLC under the followingconditions:

Column

TSK gel ODS-80TM column (4.6 mm×30 cm, manufactured by TOSOHCORPORATION)

Liquid phase

0.02 M ammonium acetate buffer (pH 4.0)

Temperature

50° C.

Flow rate

1 ml/min

Detection

Fluorescence detector (excitation wavelength 320 nm, radiationwavelength 400 nm)

Identification of the product was carried out by comparing elution timeof aminopyridine-labeled lacto-N-tetraose with that of the labeledproduct.

By the reaction, 0.17 mM (0.12 g/l) lacto-N-tetraose was formed.

EXAMPLE 20 Production of lacto-N-neotetraose

In the same manner as in Example 19, GlcNAcβ1-3Galβ1-4Glc was preparedfrom lacto-N-neotetraose and used as a complex carbohydrate precursor.

A 36 μl portion of a reaction solution containing 0.5 mM of the complexcarbohydrate precursor, 0.5 U β1,4-galactosyltransferase (manufacturedby Sigma), 6 μl of a reaction solution containing UDP-Gal (5 mM)obtained in Example 4, 100 mM Tris-HCl buffer (pH 7.9), 10 mM MnCl₂ and2 mM β-mercaptoethanol was allowed to stand for 65 hours at 32° C. toeffect the reaction.

After completion of the reaction, amount of the product accumulated inthe reaction solution was measured by HPLC under the same conditions ofExample 19-2). Identification of the product was carried out bycomparing elution time of the aminopyridine-labeled lacto-N-neotetraosewith that of the product.

By the reaction, 0.15 mM (0.11 g/l) lacto-N-neotetraose was formed.

EXAMPLE 21 Production of lacto-N-fucopentaose III

IgG Sepharose-linked α1,3-fucosyltransferase was prepared from namalwacell line KJM-1 transformed with plasmid pAMoA-FT6 (J. Biol. Chem., 269,14730 (1994)) containing a gene encoding a fusion protein of the IgGbinding region of protein A with α1,3-fucosyltransferase, in the samemanner as in Example 19-1), and used as the enzyme source ofα1,3-fucosyltransferase,

A 50 μl portion of a reaction solution containing 0.25 mMlacto-N-neotetraose (manufactured by Oxford Glycosystems), 1.0 U of theIgG Sepharose-linked α1,3-fucosyltransferase, 6 μl of a reactionsolution containing GDP-Fuc (0.25 mM) obtained in Example 16, 100 mMTris-HCl buffer (pH 7.9) and 10 mM MnCl₂ was allowed to stand for 24hours at 37° C. to effect the reaction.

After completion of the reaction, amount of the product accumulated inthe reaction solution was measured using a sugar analyzer (DX-500)manufactured by Dionex. Identification of the product was carried out bycomparing elution time of lacto-N-fucopentaose III (manufactured byOxford Glycosystems) with that of the product.

By the reaction, 0.21 mM (0.18 g/l) lacto-N-fucopentaose III was formed.

EXAMPLE 22 Construction of α1,4-galactosyltransferase (lgtC) ExpressionPlasmid

Chromosomal DNA of Neisseria gonorrhoeae (ATCC 33084) was prepared inthe same manner as in Example 1.

The sense strand DNA primer shown in SEQ ID NO:24 and the antisensestrand DNA primer shown in SEQ ID NO:25 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of Neisaeria gonorrhoeae (ATCC 33084) as the template,under the same conditions as described in the foregoing.

After completion of the PC treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes HindIII and BamHI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 1.0kb was recovered using Gene Clean II Kit. A 0.2 μg portion of the pPA31DNA obtained in Example 1-1) was cleaved with restriction enzymesHindIII and BamHI, the resulting DNA fragments separated by agarose gelelectrophoresis and then a fragment of 4.2 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 1.0 kb and 4.2 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μq/ml ampicillinand then cultured overnight at 30° C.

By extracting a plamid from the thus grown colonies of the transformantin accordance with the usual process, a lgtC expression plamid pGT3 wasobtained. Structure of the plasmid was recognized by restriction enzymecleavage (FIG. 15) .

EXAMPLE 23 Production of Globotriose

Escherichia coli NM522/pNT25/pNT32 obtained in Example 4 and Escherichiacoli NM522/pGT3 obtained in Example 22 were cultured in the same manneras in Example 2, and each of the thus obtained culture broths wascentrifuged to obtain wet cells. Also, Corynebacterium ammoniagenes ATCC21170 was cultured in the same manner as in Example 2, and the culturebroth was centrifuged to obtain wet cells. As occasion demands, thesewet cells can be preserved at −20° C. and utilized by thawing the cellsprior to use.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25/pNT32 wet cells, 50 g/l Escherichia coliNM522/pGT3 wet cells, 150 g/l Corynebacterium ammoniagenes ATCC 21170wet cells, 100 g/l fructose, 100 g/l galactose, 100 g/l lactose, 15 g/lKH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 10 g/l orotic acid(potassium salt), 4 g/l Nymeen S-215 and 10 ml/l xylene was put into a200 ml beaker, and 36 hours of the reaction was carried out at 32° C.under stirring the reaction solution with a magnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and galactose, lactose, fructose and KH₂PO₄ added whenrequired.

By the reaction, 188 g/l globotriose was formed in the reactionsolution.

Cells were removed from the reaction solution by centrifugation, and a10 ml portion of the thus obtained supernatant was purified by employinga process in which activated carbon was used, thereby obtaining 1. 5 gof globotriose as white powder.

EXAMPLE 24 Production of Galα1-4Galβ1-4GlcNAc

Escherichia coli NM522/pNT25/pNT32 obtained in Example 4 and Escherichiacoli NM522/pGT3 obtained in Example 22 were cultured in the same manneras in Example 2, and each of the thus obtained culture broths wascentrifuged to obtain wet cells. Also, Corynebacterium ammoniagenes ATCC21170 was cultured in the same manner as in Example 2, and the culturebroth was centrifuged to obtain wet cells. As occasion demands, thesewet cells can be preserved at −20° C. and utilized by thawing the cellsprior to use.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25/pNT32 wet cells, 50 g/l Escherichia coliNM522/pGT3 wet cells, 150 g/l Corynebacterium ammoniagenes ATCC 21170wet cells, 50 g/l fructose, 50 g/l galactose, 96 g/lN-acetyllactosamine, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid,10 g/l orotic acid (potassium salt), 4 g/l Nymeen S-215 and 10 ml/lxylene was put into a 200 ml beaker, and 23 hours of the reaction wascarried out at 32° C. under stirring the reaction solution with amagnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and galactose, fructose and KH₂PO₄ were added whenrequired.

By the reaction, 10 g/l Galα1-4Galβ1-4GlcNAc was formed in the reactionsolution.

Cells were removed from the reaction solution by centrifugation, and theformed product was purified from a 30 ml portion of the thus obtainedsupernatant by employing a process in which activated carbon was used,thereby obtaining 0.2 g of Galα1-4Galβ1-4GlcNAc as white powder.

EXAMPLE 25 Construction of β1,4-galactosyltransferase (lgtB) ExpressionPlasmid

The sense strand DNA primer shown in SEQ ID NO:26 and the antisencestrand DNA primer shown in SEQ ID NO:27 were synthesized. The PCR wascarried out using the synthesized DNA strands as primers, and thechromosomal DNA of N. gonorrhoeae (ATCC 33084) as the template, underthe same conditions as described in the foregoing.

After completion of the PC treatment, a precipitate of DNA was obtainedby the ethanol precipitation process. The precipitate was dissolved in20 μl of TE. Using 5 μl portion of the solution, the DNA was cleavedwith restriction enzymes HindIII and BamHI, the resulting DNA fragmentswere separated by agarose gel electrophoresis and then a fragment of 0.8kb was recovered using Gone Clean II Kit. A 0.2 μg portion ofpBluescript II SK+ DNA was cleaved with restriction enzymes HindIII andBamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a fragment of 3.0 kb was recovered in the samemanner.

Using a ligation kit, the fragments of 0.8 kb and 3.0 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured night at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, plasmid pNT60P containing the lgtBgene was obtained. Structure of the plasmid was recognized byrestriction enzyme cleavage (FIG. 16).

A 0.5 μg portion of the pNT60P DNA was cleaved with restriction enzymesClaI and BamHI, the resulting DNA fragments were separated by agarosegel electrophoresis and then a fragment of 0.8 kb was recovered. A 0.2μg portion of the pPAC31 DNA obtained in Example 1-1) was cleaved withrestriction enzymes ClaI and BamHI, the resulting DNA fragments wereseparated by arose gel electrophoresis and then a fragment of 5.5 kb wasrecovered in the same manner.

Using a ligation kit, the fragments of 0.8 kb and 5.5 kb were subjectedto ligation reaction for 16 hours at 16° C.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the usual process, and the resultingtransformant was spread on LB agar medium containing 50 μg/ml ampicillinand then cultured overnight at 30° C.

By extracting a plasmid from the thus grown colonies of the transformantin accordance with the usual process, a lgtB expression plasmid pNT60was obtained. Structure of the plasmid was recognized by restrictionenzyme cleavage (FIG. 16).

EXAMPLE 26 Production of N-acetyllactosamine

Escherichia coli NM522/pNT60 obtained in Example 25 and Escherichia coliNM522/pNT25 obtained in Example 3 were cultured in the same manner as inExample 2, and each of the thus obtained culture broths was centrifugedto obtain wet cells. Also, Corynabacterium ammoniagenes ATCC 21170 wascultured in the same manner as in Example 2, and the culture broth wascentrifuged to obtain wet cells. As occasion demands, these wet cellscan be preserved at −20° C. and utilized by thawing the cells prior touse.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25 wet cells, 50 g/l Escherichia coliNM522/pNT60 wet cells, 150 g/l Corynabacterium ammoniagenes ATCC 21170wet cells, 10 g/l orotic acid (potassium salt), 100 g/l fructose, 100g/l N-acetylglucosamine, 100 g/l galactose, 15 g/l KH₂PO₄, 5 g/lMgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylene wasput into a 200 ml beaker, and 34 hours of the reaction was carried outat 32° C. under stirring the reaction solution with a magnetic stirrer(900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and galactose, fructose and KH₂PO₄ were added whenrequired.

By the reaction, 114 g/l N-acetyllactosamine was formed in the reactionsolution.

EXAMPLE 27 Production of Lactose

Escherichia coli NM522/pNT60 obtained in Example 25 and Escherichia coliNM522/pNT25 obtained in Example 3 were cultured in the same manner as inExample 2, and each of the thus obtained culture broths was centrifugedto obtain wet cells. Also, Corynabacterium ammoniagenes ATCC 21170 wascultured in the same manner as in Example 2, and the culture broth wascentrifuged to obtain wet cells. As occasion demands, these wet cellscan be preserved at −20° C. and utilized by thawing the cells prior touse.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25 wet cells, 50 g/l Escherichia coliNM522/pNT60 wet cells, 150 g/l Corynabacterium ammoniagenes ATCC 21170wet cells, 10 g/l orotic acid (potassium salt), 115 g/l glucose, 115 g/lgalactose, 15 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/lNymeen S-215 and 10 ml/l xylene was put into a 200 ml beaker, and 15hours of the reaction was carried out at 32° C. by stirring the reactionsolution on a magnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO4 was added when required.

By the reaction, 49 g/l lactose was formed in the reaction solution.

EXAMPLE 28 Production of Globotriose

Escherichia coli NM522/pNT60 obtained in Example 25, Escherichia coliNM522/pNT25 obtained in Example 3 and Escherichia coli NM522/pGT3obtained in Example 22 were cultured in the same manner as in Example 2,and each of the thus obtained culture broths was centrifuged to obtainwet cells. Also, Corynebacterium ammoniagenes ATCC 21170 was cultured inthe same manner as in Example 2, and the culture broth was centrifugedto obtain wet cells. As occasion demands, these wet cells can bepreserved at −20° C. and utilized by thawing the cells prior to use.

A 30 ml portion of a reaction solution having a composition of 50 g/lEscherichia coli NM522/pNT25 wet cells, 50 g/l Escherichia coliNM522/pNT60 wet cells, 50 g/l Escherichia coli NM522/pGT3 wet cells, 150g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 10 g/l oroticacid (potassium salt), 115 g/l glucose, 115 g/l galactose, 15 g/lKH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10ml/l xylene was put into a 200 ml beaker, and 13 hours of the reactionwas carried out at 32° C. under stirring the reaction solution with amagnetic stirrer (900 rpm).

During the reaction, pH of the reaction solution was maintained at 7.2using 4 N NaOH, and KH₂PO₄ was added when required.

By the reaction, 5 g/l globotriose was formed in the reaction solution.

INDUSTRIAL APPLICABILITY

The present invention renders possible efficient industrial productionof a sugar nucleotide from a nucleotide precursor and a sugar as thesolo starting materials and of a complex carbohydrate from the sugarnucleotide and a complex carbohydrate precursor.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 27(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 31 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGAGAAAGCT TATGGCTGCC ATTAATACGA A         #                  #          31 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AACACGGATC CGGATGTTAC TTCTTAATGC          #                  #           30 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ATGGAGGATC CTGCTCTGTA TACCGTCT          #                  #             28 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:TGCTGGTCGA CCTGCGCTTG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AAGGAAAGCT TATGACGCAA TTTAATCCCG T         #                  #          31 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:GCAAAGTTAA CAGTCGGTAC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TCAGGAAGCT TATGTTGAAT AATGCTATGA G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:TCTCCGGATC CCATGTGACC GGGTTAG           #                  #             27 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:TCTAAATCGA TGCAGACAAA GGACAAAG          #                  #             28 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:TTGCAGGATC CTCGTAGGCC TGATAAG           #                  #             27 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:TGATATCCGC TCCCTTTCCG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 26 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:ACAGCGGATC CGATGTGTTC GCTGAG           #                  #              26 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ACAGCAAGCT TTTGACTTTA GCGGAGCAG          #                  #            29 (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GAGTTGGATC CCGATATAAA AGGAAGGAT          #                  #            29 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:TTTTTAAGCT TCATTTATCA AGAGT           #                  #               25 (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:TTTTTGATAT CCCCAATGCT GGGGGTTTTT G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs               (b)TYPE:  #nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CGTCAAAGCT TAAATGATAT TCGGGGATAA T         #                  #          31 (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:AGGGAGGATC CGACATTACT CGTTC           #                  #               25 (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 33 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:CCGCAAGATC TCGTAAAAAG GGTATCGATA AGC        #                  #         33 (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:TTGGGAAGCT TCCGGCAAAT GTGGTTT           #                  #             27 (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:ATAAACTCGA GAGAGACAAG CGGAG           #                  #               25 (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:TATTATCGAT GAATTAATAA TTCATAG           #                  #             27 (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:CTCTGGATCC AGTTACGTAT AATAT           #                  #               25 (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:CGGCAAGCTT ATTGTGCCTT TCCAATAAAA          #                  #           30 (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:ACTTGGATCC CCGTCAATAA ATCTTGCG          #                  #             28 (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:GGTAAAGCTT ATGCAAAACC ACGTTATCAG          #                  #           30 (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:AAACGGATCC TTATTGGAAA GGCACAATA          #                  #            29

What is claimed is:
 1. A process for producing guanosine diphospho-sugar(“GDP-sugar”) or uridine diphospho-sugar (“UDP-sugar”), which comprises:selecting, as enzyme sources, a) a culture of a microorganism capable ofproducing guanosine-5′-triphosphate (“GTP”) or uridine-5′-triphosphate(“UTP”) from a nucleotide precursor, or a treated product of the cultureselected from the group consisting of a concentrated product of theculture, a dried product of the culture, a culture supernatant obtainedby centrifuging the culture, a concentrated product of the culturesupernatant, an enzyme preparation obtained from the culturesupernatant, cells obtained by centrifuging the culture, a dried productof the cells, a freeze-dried product of the cells, a surfactant-treatedproduct of the cells, an ultrasonic-treated product of the cells, amechanically disrupted product of the cells, a solvent-treated productof the cells, an enzyme-treated product of the cells, a protein fractionof the cells, an immobilized product of the cells and an enzymepreparation obtained by extraction from the cells, and b) a culture orcultures of at least one strain of microorganism having genesresponsible for production of GDP-sugar or UDP-sugar from a sugarselected from the group consisting of glucose, fructose, galactose,glucosamine, N-acetylglucosamine, N-acetylgalactosamine, mannose andfucose and GTP or UTP, or a treated product of the culture selected fromthe group consisting of a concentrated product of the culture, a driedproduct of the culture, a culture supernatant obtained by centrifugingthe culture, cells obtained by centrifuging the culture, a dried productof the cells, a freeze-dried product of the cells, a surfactant-treatedproduct of the cells, a solvent-treated product of the cells, and animmobilized product of the cells wherein the treated product of theculture continues to have the same enzymatic activity as said culturecapable of producing UDP-sugar or GDP-sugar from the sugar and UTP orGTP; allowing the enzyme sources, the nucleotide precursor and the sugarto be present in an aqueous medium to form and accumulate GDP-sugar orUDP-sugar in the aqueous medium; and recovering GDP-sugar or UDP-sugarfrom the aqueous medium.
 2. The process according to claim 1, whereinthe nucleotide precursor is orotic acid, uracil, orotidine, uridine,cytosine, cytidine, adenine, adenosine, guanine, guanosine,hypoxanthine, inosine, xanthine, xanthosine, inosine-5′-monophosphate,xanthosine-5′-monophosphate, guanosine-5′-monophosphate,uridine-5′-monophosphate or cytidine-5′-monophosphate.
 3. The processaccording to claim 1, wherein the microorganism capable of producing GTPor UTP from a nucleotide precursor is a microorganism selected frommicroorganisms belonging to the genus Corynebacterium.
 4. The processaccording to claim 3, wherein the microorganism belonging to the genusCorynebacterium belongs to Corynebacterium ammoniagenes.
 5. The processaccording to claim 1, wherein the at least one strain of microorganismhaving genes responsible for production of a sugar nucleotide comprisesa recombinant microorganism having at least one gene responsible forproduction of a sugar nucleotide, said gene being derived from adifferent microorganism, or being derived from said strain ofmicroorganism but being harbored in a plasmid.
 6. The process accordingto claim 5, wherein the recombinant microorganism is selected frommicroorganisms belonging to the genus Escherichia and the genusCorynebacterium.
 7. The process according to claim 6, wherein therecombinant microorganism is Escherichia coli.
 8. The process accordingto claim 6, wherein the recombinant microorganism is Corynebacteriumammoniagenes.